Mammalian cell surface antigens

ABSTRACT

Purified genes encoding a T cell surface antigen from a mammal, reagents related thereto including purified proteins, specific antibodies, and nucleic acids encoding said antigen. Methods of using said reagents and diagnostic kits are also provided.

This is a divisional of application U.S. Ser. No. 08/348,792, filed onDec. 2, 1994 now abandoned.

FIELD OF THE INVENTION

The present invention pertains to compositions related to proteins whichfunction in controlling activation of mammalian cells, e.g., cells of amammalian immune system. In particular, it provides purified genes,proteins, antibodies, and related reagents useful, e.g., to regulateactivation, development, differentiation, and function of various celltypes, including hematopoietic cells.

BACKGROUND OF THE INVENTION

The activation of resting T cells is critical to most immune responsesand allows these cells to exert their regulatory or effectorcapabilities. See Paul (ed; 1993) Fundamental Immunology 3d ed., RavenPress, N.Y. Increased adhesion between T cells and antigen presentingcells (APC) or other forms of primary stimuli, e.g., immobilizedmonoclonal antibodies (mAb), can potentiate the T-cell receptor signals.T-cell activation and T cell expansion depends upon engagement of theT-cell receptor (TCR) and co-stimulatory signals provided by accessorycells. See, e.g., Jenkins and Johnson (1993) Curr. Opin. Immunol.5:361-367; Bierer and Hahn (1993) Semin. Immunol. 5:249-261; June, etal. (1990) Immunol. Today 11:211-216; and Jenkins (1994) Immunity1:443-446. A major, and well-studied, co-stimulatory interaction for Tcells involves either CD28 or CTLA-4 on T cells with either B7 or B70(Jenkins (1994) Immunity 1:443-446). Recent studies on CD28 deficientmice (Shahinian, et al. (1993) Science 261:609-612; Green, et al. (1994)Immunity 1:501-508) and CTLA-4 immunoglobulin expressing transgenic mice(Ronchese, et al. (1994) J. Exp. Med. 179:809-817) have revealeddeficiencies in some T-cell responses though these mice have normalprimary immune responses and normal CTL responses to lymphocyticchoriomeningitis virus and vesicular stomatitis virus. As a result, boththese studies conclude that other co-stimulatory molecules must besupporting T-cell function. However, identification of these moleculeswhich mediate distinct costimulatory signals has been difficult.

The inability to modulate activation signals prevents control ofinappropriate developmental or physiological responses in the immunesystem. The present invention provides at least one alternativecostimulatory molecule, agonists and antagonists of which will be usefulin modulating a plethora of immune responses.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the discovery of anantigen which acts as a costimulator of T cell activation. Inparticular, it provides a gene encoding a glycosylated 70 kDa protein,designated SLAM, which is expressed on CD4⁺, CD8⁺ thymocytes andperipheral blood CD45RO^(high) memory T cells, and is rapidly induced onnaive T cells following activation. Engagement of SLAM directlystimulates proliferation of CD4⁺ T cell clones and enhancesantigen-specific proliferation and cytokine production by CD4⁺ T cells.Particularly the production of IFN-γ is strongly upregulated, even in Thelper type 2 (Th2) CD4⁺ T cell clones, whereas no induction of IL-4 orIL-5 production was observed in Th1 clones. These data indicate SLAM isa novel T-cell co-stimulatory molecule which, when engaged, potentiatesT cell expansion and induces a Th0/Th1 cytokine production profile. Bothhuman and mouse embodiments are described, enabling mammalian genes,proteins, antibodies, and uses thereof. Functional equivalentsexhibiting significant sequence homology are available fromnon-mammalian species. Moreover, SLAM can function as its bindingpartner to stimulate other cells expressing the antigen in a homophilicinteraction.

More particularly, the present invention provides a substantially pureor recombinant SLAM protein or peptide fragment thereof. Variousembodiments include a protein or peptide selected from a protein orpeptide from a warm blooded animal selected from the group of birds andmammals, including a human or mouse; a protein or peptide comprising atleast one polypeptide segment of SEQ ID NO: 2, 4, 6, 8, 10, or 12; aprotein or peptide which exhibits a post-translational modificationpattern distinct from natural SLAM; or a protein or peptide which iscapable of co-stimulating a T cell with another signal. The protein orpeptide can comprise a sequence from the extracellular or theintracellular portion of a SLAM; or be a fusion protein. Anotherembodiment is a composition comprising a SLAM protein and apharmaceutically acceptable carrier.

The invention also embraces an antibody which specifically binds a SLAMprotein or peptide, e.g., wherein the SLAM is a mammalian protein,including a human or mouse; the antibody is raised against a purifiedSLAM peptide sequence of SEQ ID NO; 2, 4, 6, 8, 10, or 12; the antibodyis a monoclonal antibody; or the antibody is labeled. The antibodiesalso make available a method of purifying a SLAM protein or peptide fromother materials in a mixture comprising contacting the mixture to ananti-SLAM antibody, and separating bound SLAM from other materials.

Another aspect of the invention is an isolated or recombinant nucleicacid capable of encoding a SLAM protein or peptide, including a nucleicacid which encodes a sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12; whichincludes a sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 11; which encodes asequence from an extracellular domain of a natural SLAM; or whichencodes a sequence from an intracellular domain of a natural SLAM. Suchnucleic acid embodiments also include an expression or replicatingvector.

The invention also provides a kit containing a substantially pure SLAMor fragment; an antibody or receptor which specifically binds a SLAM; ora nucleic acid, or its complement, encoding a SLAM or peptide. This kitalso provides methods for detecting in a sample the presence of anucleic acid, protein, or antibody, comprising testing said sample withsuch a kit.

The invention also supplies methods of modulating the physiology of acell comprising contacting said cell with a substantially pure SLAM orfragment; an antibody or binding partner which specifically binds aSLAM; or a nucleic acid encoding a SLAM or peptide. Certain preferredembodiments include a method where the cell is a T cell and themodulating of physiology is activation of the T cell; or where the cellis in a tissue and/or in an organism.

Also provided are a method of expressing a SLAM peptide by expressing anucleic acid encoding a SLAM polypeptide. The invention also provides acell, tissue, organ, or organism comprising a nucleic acid encoding aSLAM peptide.

The invention also provides a recombinant nucleic acid comprisingsequence at least about 70% identity over a stretch of at least about 30nucleotides to a SLAM nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9,or 11, useful, e.g., as a probe or PCR primer for a related gene.Another embodiment encodes a polypeptide comprising at least about 60%identity over a stretch of at least about 20 amino acids to a SLAMsequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

All references cited herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

OUTLINE

I. General

II. Purified SLAM

A. physical properties

B. biological properties

III. Physical variants

A. sequence variants, fragments

B. post-translational variants

1. glycosylation

2. others

IV. Functional Variants

A. analogs, fragments

1. agonists

2. antagonists

B. mimetics

1. protein

2. chemicals

C. species variants

V. Antibodies

A. polyclonal

B. monoclonal

C. fragments, binding compositions,

VI. Nucleic Acids

A. natural isolates; methods

B. synthetic genes

C. methods to isolate

VII. Making SLAM, mimetics

A. recombinant methods

B. synthetic methods

C. natural purification

VIII. Uses

A. diagnostic

B. therapeutic

IX. Kits

A. nucleic acid reagents

B. protein reagents

C. antibody reagents

I. General

The present invention provides amino acid sequences and DNA sequencesencoding various mammalian proteins which are antigens found in theearly stages of T cell activation, e.g., which can activate a T cell.Among these proteins are antigens which induce proliferation of T cells,among other physiological effects. The full length antigens, andfragments, will be useful in both physiological modulation of cellsexpressing the antigen. The proteins will also be useful as antigens,e.g., immunogens, for raising antibodies to various epitopes on theprotein, both linear and conformational epitopes.

Monoclonal antibodies (mAb) were raised to molecules expressed in theearly phase of T-cell activation. One antibody designated A12 had uniqueagonistic effects on T cell clones and recognized a previouslyunidentified early activation molecule designated SLAM. A12 directlyinduced proliferation of CD4⁺ T cell clones belonging to the Th0, Th1,and Th2-like subsets. In the absence of any other stimuli, A12 or itsF(Ab')₂ induced proliferation of T cell clones B21, ChT38, HY06, andTA23, whereas consistent with previous studies, see June, et al. (1990)Immunol. Today 11:211-216, engagement of CD28 was ineffective. Thesedata indicate that SLAM acts independently of CD28 and that it plays anovel and important role in T cell activation.

A cDNA encoding SLAM was isolated from a T-cell cDNA library byexpression cloning using A12 for selection. The SLAM cDNA was 1860 bp inlength and contained one large open reading frame encoding a type Itransmembrane protein with a 27 amino-acid N-terminal hydrophobic leadersequence, a 202 amino-acid extracellular region which contains 8potential N-glycosylation sites, a 22 amino-acid hydrophobic membranespanning portion, and a 77 amino-acid cytoplasmic domain. See SEQ. ID.NO: 1. Three of the four potential tyr phosphorylation sites in thecytoplasmic domain of SLAM conform to the consensus sequencephosphotyrosine-hydrophobic-X-hydrophobic, determined for binding to oneclass of SH2 domains. See zhou, et al. (1993) Cell 72:767-778. Antiseraraised against recombinant SLAM precipitated a 70 kD glycoprotein froman activated CD4⁺ T-cell clone. N-glycanase treatment of the SLAMimmunoprecipitate revealed a protein core of 40 kDa, which correlateswith the predicted molecular size. SLAM exhibits characteristics of amember of the immunoglobulin (Ig) supergene family, with one variableand one constant domain, and shows some degree of homology with CD48(26% homology; see Staunton and Thorley-Lawson (1987) EMBO J.6:3695-3701), LFA-3/CD58 (17% homology; see Seed (1987) Nature329:840-842), and a recently cloned signaling molecule expressed onmurine NK and cytotoxic T cells called 2B4 (28% homology; see Mathew, etal. (1993) J. Immunol. 151:5328-5337).

Using PCR to detect transcripts in various tissues and cell types, it isclear that SLAM is expressed primarily in lymphoid cells. Activatedperipheral blood mononuclear cells (PBMC) contain a 1.9 kb transcript,corresponding to the size of the cloned SLAM cDNA and also a 4 kbtranscript. The 4 kb mRNA is composed of at least two differenttranscripts, including one encoding a secreted form of SLAM lacking 30amino-acids, including the entire 22 amino-acid transmembrane region,and another which encodes transmembrane SLAM. An alternatively spliced 2kb cDNA clone was also identified, encoding a form of SLAM with atruncated cytoplasmic domain.

SLAM mRNA is induced within 2 h after activation, which correlates withits rapid appearance on the T-cell surface. SLAM is not expressed onCD45RA⁺ naive T cells, but can be detected at low levels onCD45RO^(high) memory T cells in the absence of in vitro activation. SLAMexpression is rapidly induced (within 3 h) on naive CD45RA⁺ T cells andenhanced on CD45RO^(high) T cells following activation, and maximalexpression occurs at 6-8 h. Immature CD3^(low), CD4⁺, CD8⁺ fetalthymocytes express SLAM, whereas the more mature CD3^(high) single CD4⁺or CD8⁺ thymocytes are mostly negative. SLAM is expressed at very lowlevels on peripheral B cells and is upregulated with activation but isnot present on monocytes.

The presence of SLAM on B cells and CD45RO^(high) memory T cells, andthe natural occurrence of a soluble form of SLAM, suggest a broadfunction of this molecule. The findings that co-stimulation via SLAMenhances Ag-specific proliferative responses and induces Th0/Th1cytokine production profiles in T cell clones, including Th2 clones,suggests that the interaction between SLAM and its ligand willcontribute to T cell expansion and the generation of Th0 or Th1responses.

In addition to its direct stimulatory effects on T cell clones, SLAMacts as a co-stimulatory molecule for T-cell activation. The optimalantigen-specific proliferative responses of peripheral blood T cells ofdonors immunized with tetanus toxoid (TT) or purified protein derivative(PPD) were further enhanced in a dose dependent fashion by the additionof A12 F(ab')₂, indicating that specific engagement of SLAM isresponsible for the enhanced T-cell responses. Generally a 2-3 foldincrease in proliferation was observed. Similarly, the optimalantigen-specific proliferation of CD4⁺ T-cell clones were enhanced inthe presence of A12 or A12 F(ab')₂ in a dose-dependent manner. Thisenhancement was observed with CD4⁺ T cell clones belonging to the Th2,Th0, and Th1 subsets. The co-stimulatory effects mediated through SLAMon T cells were not restricted to Ag-specific stimulation, as T-cellproliferation induced by anti-CD3 mAb was also enhanced by A12. Even atoptimal anti-CD3 concentrations, a further 2-3-fold increase in theproliferation was observed upon engagement of SLAM by A12.

Cytokine production by a panel of CD4⁺ T-cell clones belonging todifferent subsets stimulated by their respective antigens wasupregulated following SLAM engagement by A12. In particular, IFN-γproduction was strongly enhanced by A12 and A12 F(Ab')₂.

Co-stimulation of Th2 clones with A12 or its F(Ab')₂ stronglyupregulated (5-17 fold) IFN-γ production, whereas there were little(less than 2 fold), or no, enhancing effects on IL-4 production by fourclones tested. The levels of IFN-γ production induced in the presence ofA12 by Th2 clones were comparable to those induced by antigen in Th1 andTh0 clones. A12 co-stimulation also preferentially enhanced IFN-γproduction by Th0 and Th1 clones. In contrast to its strongIFN-γ-inducing effects on Th2 clones, costimulation via SLAM did notinduce IL-4 or IL-5 production by Th1 clones.

These results indicate that T cell co-stimulation via SLAM results in apreferential induction of IFN-γ production, even in allergen-specificCD4⁺ T-cell clones of the Th2-subset, thereby reversing the phenotype ofthese cells to a clear Th0 cytokine production profile. The cytokineproduction pattern defining established Th1 clones, however, is notaltered by co-stimulation via SLAM.

In order to identify the natural ligand for SLAM, a SLAM-immunoglobulinfusion protein (SLAM-Ig) was generated. The SLAM portion of SLAM-Igbound specifically to L cells stably transfected with SLAM. In addition,SLAM-Ig interacted homophilically in solution demonstrating that SLAMcan serve as a self-ligand. SLAM-Ig binding to various cell types alsocorrelated with their SLAM expression. Unlike other described ligandsfor T cells, SLAM expressed on L cells provided a direct proliferativesignal for human T cell clones in the absence of any other stimuli. Thisnovel stimulatory activity provided by homophilic interaction of SLAMwas resistant to cyclosporin.

                                      TABLE 1                                     __________________________________________________________________________    Human SLAM Sequences.                                                         __________________________________________________________________________    Human SLAM1 (pSURslam1) nucleotide and predicted amino acid                    sequence. Predicted leader sequence and the transmembrane                     sequence are underlined, though natural boundaries may be                     different, also depending on cell type. An exon encoding                      the transmembrane domain which is not present in human SLAM3                  (pSECslam) is delineated by two •s and the bases bordering              this exon are in bold type (nucleotides numbered 761 and                      850). Cysteines are found at amino acid residues numbered                     32, 132, 158, 164, 209, 228, and 303. Potential N-linked                      glycosylation sites are found at residues numbered 53, 57,                    102, 125, 150, 155, 189, and 217. Fragments between cysteines                 and/or N-linked glycosylation sites are particularly useful                   in generating antibodies.  SEQ ID NO: 1 and 2.                               __________________________________________________________________________      -                                                                                   10        20        30        40        50        60                   aggcatctgtgagcagctgccaggctccggccaggatcccttccttctcctcattggctg                   -         70        80        90       100       110       120               atggatcccaaggggctcctctccttgaccttcgtgctgtttctctccctggcttttggg                   M  D  P  K  G  L  L  S  L  T  F  V  L  F  L  S  L  A  F  G                   1                                                                              -        130       140       150       160       170       180               gcaagctacggaacaggtgggcgcatgatgaactgcccaaagattctccggcagttggga                   A  S  Y  G  T  G  G  R  M  M  N  C  P  K  I  L  R  Q  L  G                   21                                                                             -        190       200       210       220       230       240               agcaaagtgctgctgcccctgacatatgaaaggataaataagagcatgaacaaaagcatc                   S  K  V  L  L  P  L  T  Y  E  R  I  N  K  S  M  N  K  S  I                   41                                                                             -        250       260       270       280       290       300               cacattgtcgtcacaatggcaaaatcactggagaacagtgtcgagaacaaaatagtgtct                   H  I  V  V  T  M  A  K  S  L  E  N  S  V  E  N  K  I  V  S                   61                                                                             -        310       320       330       340       350       360               cttgatccatccgaagcaggccctccacgttatctaggagatcgctacaagttttatctg                   L  D  P  S  E  A  G  P  P  R  Y  L  G  D  R  Y  K  F  Y  L                   81                                                                             -        370       380       390       400       410       420               gagaatctcaccctggggatacgggaaagcaggaaggaggatgagggatggtaccttatg                   E  N  L  T  L  G  I  R  E  S  R  K  E  D  E  G  W  Y  L  M                   101                                                                            -        430       440       450       460       470       480               accctggagaaaaatgtttcagttcagcgcttttgcctgcagttgaggctttatgagcag                   T  L  E  K  N  V  S  V  Q  R  F  C  L  Q  L  R  L  Y  E  Q                   121                                                                            -        490       500       510       520       530       540               gtctccactccagaaattaaagttttaaacaagacccaggagaacgggacctgcaccttg                   V  S  T  P  E  I  K  V  L  N  K  T  Q  E  N  G  T  C  T  L                   141                                                                            -        550       560       570       580       590       600               atactgggctgcacagtggagaagggggaccatgtggcttacagctggagtgaaaaggcg                   I  L  G  C  T  V  E  K  G  D  H  V  A  Y  S  W  S  E  K  A                   161                                                                            -        610       620       630       640       650       660               ggcacccacccactgaacccagccaacagctcccacctcctgtccctcaccctcggcccc                   G  T  H  P  L  N  P  A  N  S  S  H  L  L  S  L  T  L  G  P                   181                                                                            -        670       680       690       700       710       720               cagcatgctgacaatatctacatctgcaccgtgagcaaccctatcagcaacaattcccag                   Q  H  A  D  N  I  Y  I  C  T  V  S  N  P  I  S  N  N  S  Q                   201                                                                            -        730       740       750       760•     770       780          accttcagcccgtggcccggatgcaggacagacccctcagaaacaaaaccatgggcagtg                   T  F  S  P  W  P  G  C  R  T  D  P  S  E  T  K  P  W  A  V                   221                                                                            -        790       800       810       820       830       840               tatgctgggctgttagggggtgtcatcatgattctcatcatggtggtaatactacagttg                   Y  A  G  L  L  G  G  V  I  M  I  L  I  M  V  V  I  L  Q  L                   241                                                                            -          •       860       870       880       890       900         agaagaagaggtaaaacgaaccattaccagacaacagtggaaaaaaaaagccttacgatc                   R  R  R  G  K  T  N  H  Y  Q  T  T  V  E  K  K  S  L  T  I                   261                                                                            -        910       920       930       940       950       960               tatgcccaagtccagaaaccaggtcctcttcagaagaaacttgactccttcccagctcag                   Y  A  Q  V  Q  K  P  G  P  L  Q  K  K  L  D  S  F  P  A  Q                   281                                                                            -        970       980       990      1000      1010      1020               gacccttgcaccaccatatatgttgctgccacagagcctgtcccagagtctgtccaggaa                   D  P  C  T  T  I  Y  V  A  A  T  E  P  V  P  E  S  V  Q  E                   301                                                                            -       1030      1040      1050      1060      1070      1080               acaaattccatcacagtctatgctagtgtgacacttccagagagctgacaccagagacca                   T  N  S  I  T  V  Y  A  S  V  T  L  P  E  S                                  321                                                                            -       1090      1100      1110      1120      1130      1140               acaaagggactttctgaaggaaaatggaaaaaccaaaatgaacactgaacttggccacag                   -       1150      1160      1170      1180      1190      1200               gcccaagtttcctctggcagacatgctgcacgtctgtacccttctcagatcaactccctg                   -       1210      1220      1230      1240      1250      1260               gtgatgtttcttccacatacatctgtgaaatgaacaaggaagtgaggcttcccaagaatt                   -       1270      1280      1290      1300      1310      1320               tagcttgctgtgcagtggctgcaggcgcagaacagagcgttacttgataacagcgttcca                   -       1330      1340      1350      1360      1370      1380               tctttgtgttgtagcagatgaaatggacagtaatgtgagttcagactttgggcatcttgc                   -       1390      1400      1410      1420      1430      1440               tcttggctggaactgataataaaaatcagactgaaagccaggacatctgagtacctatct                   -       1450      1460      1470      1480      1490      1500               cacacactgaccaccagtcacaaagtctggaaaagtttacattttggctatctttacttt                   -       1510      1520      1530      1540      1550      1560               gttctgggagctgatcatgataacctgcagacctgatcaagcctctgtgcctcagtttct                   -       1570      1580      1590      1600      1610      1620               ctctcaggataaagagtgaatagaggccgaagggtgaatttcttattatacataaaacac                   -       1630      1640      1650      1660      1670      1680               tctgatattattgtataaaggaagctaagaatattattttatttgcaaaacccagaagct                   -       1690      1700      1710                                             aaaaagtcaataaacagaaagaatgattttgagaaa                                         __________________________________________________________________________    Human SLAM2 (pSURslam2) nucleotide and predicted amino acid                    sequence. The human SLAM2 apparently differs from human                       SLAM1 by a differential splicing event resulting in a                         different C-terminal sequence beginning at the point indicated                by • (nucleotide 924). SEQ ID NO: 3 AND 4.                             __________________________________________________________________________      -                                                                                   10        20        30        40        50        60                   aggcatctgtgagcagctgccaggctccggccaggatcccttccttctcctcattggctg                   -         70        80        90       100       110       120               atggatcccaaggggctcctctccttgaccttcgtgctgtttctctccctggcttttggg                   M  D  P  K  G  L  L  S  L  T  F  V  L  F  L  S  L  A  F  G                   1                                                                              -        130       140       150       160       170       180               gcaagctacggaacaggtgggcgcatgatgaactgcccaaagattctccggcagttggga                   A  S  Y  G  T  G  G  R  M  M  N  C  P  K  I  L  R  Q  L  G                   21                                                                             -        190       200       210       220       230       240               agcaaagtgctgctgcccctgacatatgaaaggataaataagagcatgaacaaaagcatc                   S  K  V  L  L  P  L  T  Y  E  R  I  N  K  S  M  N  K  S  I                   41                                                                             -        250       260       270       280       290       300               cacattgtcgtcacaatggcaaaatcactggagaacagtgtcgagaacaaaatagtgtct                   H  I  V  V  T  M  A  K  S  L  E  N  S  V  E  N  K  I  V  S                   61                                                                             -        310       320       330       340       350       360               cttgatccatccgaagcaggccctccacgttatctaggagatcgctacaagttttatctg                   L  D  P  S  E  A  G  P  P  R  Y  L  G  D  R  Y  K  F  Y  L                   81                                                                             -        370       380       390       400       410       420               gagaatctcaccctggggatacgggaaagcaggaaggaggatgagggatggtaccttatg                   E  N  L  T  L  G  I  R  E  S  R  K  E  D  E  G  W  Y  L  M                   101                                                                            -        430       440       450       460       470       480               accctggagaaaaatgtttcagttcagcgcttttgcctgcagttgaggctttatgagcag                   T  L  E  K  N  V  S  V  Q  R  F  C  L  Q  L  R  L  Y  E  Q                   121                                                                            -        490       500       510       520       530       540               gtctccactccagaaattaaagttttaaacaagacccaggagaacgggacctgcaccttg                   V  S  T  P  E  I  K  V  L  N  K  T  Q  E  N  G  T  C  T  L                   141                                                                            -        550       560       570       580       590       600               atactgggctgcacagtggagaagggggaccatgtggcttacagctggagtgaaaaggcg                   I  L  G  C  T  V  E  K  G  D  H  V  A  Y  S  W  S  E  K  A                   161                                                                            -        610       620       630       640       650       660               ggcacccacccactgaacccagccaacagctcccacctcctgtccctcaccctcggcccc                   G  T  H  P  L  N  P  A  N  S  S  H  L  L  S  L  T  L  G  P                   181                                                                            -        670       680       690       700       710       720               cagcatgctgacaatatctacatctgcaccgtgagcaaccctatcagcaacaattcccag                   Q  H  A  D  N  I  Y  I  C  T  V  S  N  P  I  S  N  N  S  Q                   201                                                                            -        730       740       750       760       770       780               accttcagcccgtggcccggatgcaggacagacccctcagaaacaaaaccatgggcagtg                   T  F  S  P  W  P  G  C  R  T  D  P  S  E  T  K  P  W  A  V                   221                                                                            -        790       800       810       820       830       840               tatgctgggctgttagggggtgtcatcatgattctcatcatggtggtaatactacagttg                   Y  A  G  L  L  G  G  V  I  M  I  L  I  M  V  V  I  L  Q  L                   241                                                                            -        850       860       870       880       890       900               agaagaagaggtaaaacgaaccattaccagacaacagtggaaaaaaaaagccttacgatc                   R  R  R  G  K  T  N  H  Y  Q  T  T  V  E  K  K  S  L  T  I                   261                                                                            -        910       920  •    930       940       950       960         tatgcccaagtccagaaaccaggtgacactcatcatcagacttcggacttattctaatcc                   Y  A  Q  V  Q  K  P  G  D  T  H  H  Q  T  S  D  L  F                         281                                                                            -        970       980       990      1000      1010      1020               aggatgaccttattttgaaatccttatcttgacatctgtgaagacctttattcaaataaa                   -       1030      1040      1050      1060      1070      1080               gtcacattttgacattctgcgaggggctggagccgggccggggcgatgtggagcgcgggc                   -       1090      1100      1110      1120      1130      1140               cgcggcggggctgcctggccggtgctgttggggctgctgctggcgctgttagtgccgggc                   -       1150      1160      1170      1180      1190      1200               ggtggtgccgccaagaccggtgcggagctcgtgactgcgggtcggtgctgaagctgctca                   -       1210      1220      1230      1240      1250      1260               atacgcaccaccggtgcggctgcactcgcacgacatcaaatacggatccggcagcggcca                   -       1270      1280      1290      1300      1310      1320               gcaatcggtgaccggcgtagaggtcggagcgacgaatagctactggcggatccgcggcgg                   -       1330      1340      1350      1360      1370      1380               ctcggaggggggtgcccgcgcgggtccccggtgcgctgcgggcaggcggtgaggtcacac                   -       1390      1400      1410      1420      1430      1440               atgtgcttacgggcaagaacctgcacacgcaccacttcccgtcgccgctgtccaacaacc                   -       1450      1460      1470      1480      1490      1500               aggaagtgagtgccaaaggggaagacggcgagggcgacgacctggacctatggacagtgc                   -       1510      1520      1530      1540      1550      1560               gctgctctgctctggacagcactgggagcgtgaggctgctgtggcgccttccagcatgtg                   -       1570      1580      1590      1600      1610      1620               gcacctctgtggttcctgtcagtcacggtagcagtatggaagccccatccgtgggcagca                   -       1630      1640      1650      1660      1670      1680               tgaggtccacgcatgcccagtgccaacacgcacaatacgtggaaggccatggaaggcatc                   -       1690      1700      1710      1720      1730      1740               ttcatcaagcctagtgtggagccctctgcaggtcacgatgaactctgagtgtgtggatgg                   -       1750      1760      1770      1780      1790      1800               atgggtggatggagggtggcaggtggggcgtctgcagggccactcttggcagagactttg                   -       1810      1820      1830      1840      1850                         ggtttgtaggggtcctcaagtgcctttgtgattaaagaatgttggtctatga                         __________________________________________________________________________      -                                                                           Human SLAM3 (pSECslam) nucleotide and predicted amino acid                     sequence. The splice junction where the transmembrane domain                  sequence of SLAM1 was deleted is indicated by • (nucleotide             761). SLAM3 is secreted by COS cells transfected with                         pSECslam, confirming that SLAM3 encodes a soluble form of                     SLAM for RT-PCR, the SLAM3 transcript has been detected in                    different cell types, confirming that it is a bonafide MRNA.                  SEQ ID NO: 5 and 6.                                                          __________________________________________________________________________      -                                                                                   10        20        30        40        50        60                   aggcatctgtgagcagctgccaggctccggccaggatcccttccttctcctcattggctg                   -         70        80        90       100       110       120               atggatcccaaggggctcctctccttgaccttcgtgctgtttctctccctggcttttggg                   M  D  P  K  G  L  L  S  L  T  F  V  L  F  L  S  L  A  F  G                   1                                                                              -        130       140       150       160       170       180               gcaagctacggaacaggtgggcgcatgatgaactgcccaaagattctccggcagttggga                   A  S  Y  G  T  G  G  R  M  M  N  C  P  K  I  L  R  Q  L  G                   21                                                                             -        190       200       210       220       230       240               agcaaagtgctgctgcccctgacatatgaaaggataaataagagcatgaacaaaagcatc                   S  K  V  L  L  P  L  T  Y  E  R  I  N  K  S  M  N  K  S  I                   41                                                                             -        250       260       270       280       290       300               cacattgtcgtcacaatggcaaaatcactggagaacagtgtcgagaacaaaatagtgtct                   H  I  V  V  T  M  A  K  S  L  E  N  S  V  E  N  K  I  V  S                   61                                                                             -        310       320       330       340       350       360               cttgatccatccgaagcaggccctccacgttatctaggagatcgctacaagttttatctg                   L  D  P  S  E  A  G  P  P  R  Y  L  G  D  R  Y  K  F  Y  L                   81                                                                             -        370       380       390       400       410       420               gagaatctcaccctggggatacgggaaagcaggaaggaggatgagggatggtaccttatg                   E  N  L  T  L  G  I  R  E  S  R  K  E  D  E  G  W  Y  L  M                   101                                                                            -        430       440       450       460       470       480               accctggagaaaaatgtttcagttcagcgcttttgcctgcagttgaggctttatgagcag                   T  L  E  K  N  V  S  V  Q  R  F  C  L  Q  L  R  L  Y  E  Q                   121                                                                            -        490       500       510       520       530       540               gtctccactccagaaattaaagttttaaacaagacccaggagaacgggacctgcaccttg                   V  S  T  P  E  I  K  V  L  N  K  T  Q  E  N  G  T  C  T  L                   141                                                                            -        550       560       570       580       590       600               atactgggctgcacagtggagaagggggaccatgtggcttacagctggagtgaaaaggcg                   I  L  G  C  T  V  E  K  G  D  H  V  A  Y  S  W  S  E  K  A                   161                                                                            -        610       620       630       640       650       660               ggcacccacccactgaacccagccaacagctcccacctcctgtccctcaccctcggcccc                   G  T  H  P  L  N  P  A  N  S  S  H  L  L  S  L  T  L  G  P                   181                                                                            -        670       680       690       700       710       720               cagcatgctgacaatatctacatctgcaccgtgagcaaccctatcagcaacaattcccag                   Q  H  A  D  N  I  Y  I  C  T  V  S  N  P  I  S  N  N  S  Q                   201                                                                            -        730       740       750          •      770       780         accttcagcccgtggcccggatgcaggacagacccctcaggtaaaacgaaccattaccag                   T  F  S  P  W  P  G  C  R  T  D  P  S  G  K  T  N  H  Y  Q                   221                                                                            -        790       800       810       820       830       840               acaacagtggaaaaaaaaagccttacgatctatgcccaagtccagaaaccaggtcctctt                   T  T  V  E  K  K  S  L  T  I  Y  A  Q  V  Q  K  P  G  P  L                   241                                                                            -        850       860       870       880       890       900               cagaagaaacttgactccttcccagctcaggacccttgcaccaccatatatgttgctgcc                   Q  K  K  L  D  S  F  P  A  Q  D  P  C  T  T  I  Y  V  A  A                   261                                                                            -        910       920       930       940       950       960               acagagcctgtcccagagtctgtccaggaaacaaattccatcacagtctatgctagtgtg                   T  E  P  V  P  E  S  V  Q  E  T  N  S  I  T  V  Y  A  S  V                   281                                                                            -        970       980       990      1000      1010      1020               acacttccagagagctgacaccagagaccaacaaagggactttctgaaggaaaatggaaa                   T  L  P  E  S                                                                301                                                                          __________________________________________________________________________      -                                                                           Human SLAM4 (pCYTslam) nucleotide and predicted amino acid                     sequence. The point before which the sequence of SLAM4                        differs from SLAM1 is indicated by • (nucleotide 145) and               the base in bold type. The presence of this alternate exon                    at the 5' end predicts that SLAM4 lacks a leader sequence.                    The SLAM4 molecule when expressed in COS cells, is not                        effectively transferred to the cell surface and is                            presumably cytoplasmic. Using a 5' primer specific for the                    untranslated 5' exon of SLAM4 and a 3' primer specific for                    the SLAM coding region for RT-PCR, this transcript has been                   detected in different cell types, confirming that it a                        bonafide mRNA.  SEQ ID NO: 7 and 8.                                          __________________________________________________________________________      -                                                                                   10        20        30        40        50        60                   ggactctgttcctgtctttctgtctatcttcttcccaaggcaggctattgctttctgttt                   -         70        80        90       100       110       120               agaagtatcagggctatgagaaaaggtatttgagaaagaaaaagccaagcaaggaagtgg                   -        130       140    •  150       160       170       180         actttggactgcctgtgtgagtggggtgggcgcatgatgaactgcccaaagattctccgg                                                    M  M  N  C  P  K  I  L  R                    -        190       200       210       220       230       240               cagttgggaagcaaagtgctgctgcccctgacatatgaaaggataaataagagcatgaac                   Q  L  G  S  K  V  L  L  P  L  T  Y  E  R  I  N  K  S  M  N                   10                                                                             -        250       260       270       280       290       300               aaaagcatccacattgtcgtcacaatggcaaaatcactggagaacagtgtcgagaacaaa                   K  S  I  H  I  V  V  T  M  A  K  S  L  E  N  S  V  E  N  K                   30                                                                             -        310       320       330       340       350       360               atagtgtctcttgatccatccgaagcaggccctccacgttatctaggagatcgctacaag                   I  V  S  L  D  P  S  E  A  G  P  P  R  Y  L  G  D  R  Y  K                   50                                                                             -        370       380       390       400       410       420               ttttatctggagaatctcaccctggggatacgggaaagcaggaaggaggatgagggatgg                   F  Y  L  E  N  L  T  L  G  I  R  E  S  R  K  E  D  E  G  W                   70                                                                             -        430       440       450       460       470       480               taccttatgaccctggagaaaaatgtttcagttcagcgcttttgcctgcagttgaggctt                   Y  L  M  T  L  E  K  N  V  S  V  Q  R  F  C  L  Q  L  R  L                   90                                                                             -        490       500       510       520       530       540               tatgagcaggtctccactccagaaattaaagttttaaacaagacccaggagaacgggacc                   Y  E  Q  V  S  T  P  E  I  K  V  L  N  K  T  Q  E  N  G  T                   110                                                                            -        550       560       570       580       590       600               tgcaccttgatactgggctgcacagtggagaagggggaccatgtggcttacagctggagt                   C  T  L  I  L  G  C  T  V  E  K  G  D  H  V  A  Y  S  W  S                   130                                                                            -        610       620       630       640       650       660               gaaaaggcgggcacccacccactgaacccagccaacagctcccacctcctgtccctcacc                   E  K  A  G  T  H  P  L  N  P  A  N  S  S  H  L  L  S  L  T                   150                                                                            -        670       680       690       700       710       720               ctcggcccccagcatgctgacaatatctacatctgcaccgtgagcaaccctatcagcaac                   L  G  P  Q  H  A  D  N  I  Y  I  C  T  V  S  N  P  I  S  N                   170                                                                            -        730       740       750       760       770       780               aattcccagaccttcagcccgtggcccggatgcaggacagacccctcagaaacaaaacca                   N  S  Q  T  F  S  P  W  P  G  C  R  T  D  P  S  E  T  K  P                   190                                                                            -        790       800       810       820       830       840               tgggcagtgtatgctgggctgttagggggtgtcatcatgattctcatcatggtggtaata                   W  A  V  Y  A  G  L  L  G  G  V  I  M  I  L  I  M  V  V  I                   210                                                                            -        850       860       870       880       890       900               ctacagttgagaagaagaggtaaaacgaaccattaccagacaacagtggaaaaaaaaagc                   L  Q  L  R  R  R  G  K  T  N  H  Y  Q  T  T  V  E  K  K  S                   230 -  -        910       920       930       940       950       960         cttacgatctatgcccaagtccagaaaccaggtcctcttcagaagaaacttgactccttc                   L  T  I  Y  A  Q  V  Q  K  P  G  P  L  Q  K  K  L  D  S  F                    -        970       980       990      1000      1010      1020               ccagctcaggacccttgcaccaccatatatgttgctgccacagagcctgtcccagagtct                   P  A  Q  D  P  C  T  T  I  Y  V  A  A  T  E  P  V  P  E  S                    -       1030      1040      1050      1060      1070      1080               gtccaggaaacaaattccatcacagtctatgctagtgtgacacttccagagagctgacac                   V  Q  E  T  N  S  I  T  V  Y  A  S  V  Y  L  P  E  S                        __________________________________________________________________________      ?

The nucleotide and predicted amino acid sequence of mouse SLAM is shownin Table 2. One version of mouse SLAM is a type I transmembrane proteincontaining 9 potential N-linked glycosylation sites. The predictedunglycosylated MW is 40,000. The sequence shown is for mouse SLAM1 (inthe plasmid pMSLAM1) which is the most abundant 1.8 kb SLAM cDNA,however, another 1.8 kb cDNA SLAM2 (in pMSLAM2), representing about 25%of the cDNA's was also isolated. SLAM2 shares about the first 1 kb ofsequence with the SLAM1 sequence, but has different sequence at its 3'end. This SLAM2 cDNA in pMSLAM2 encodes a SLAM protein with a differentcytoplasmic domain. The sequence of SLAM2 cDNA is shown in Table 2 andthe position after which SLAM2 sequence varies from SLAM1 is indicated.Table 3 shows an alignment of selected human and mouse SLAM proteinsequences. As is the case for human SLAM, mouse SLAM typically has one Vand one C immunoglobulin domain and shares extensive amino-acid homologywith human SLAM over the entire molecule, this being 88% countingconservative substitutions. The homology at the nucleotide level isabout 70%. This mouse protein contains eight separate amino acidinsertions relative to that human SLAM. The cysteines in theextracellular domain are all conserved and the context of threetyrosines in the cytoplasmic domain are perfectly retained. The twodistal tyrosines in the cytoplasmic domain are not present in thealternatively spliced mouse SLAM2 molecule encoded by pSLAM2 (Table 2)and the unique portion of this cytoplasmic domain does not share highhomology with human SLAM. There is an alternatively spliced form ofhuman SLAM with a different cytoplasmic tail. The alternate sequence inpMSLAM2 is not homologous to the unique sequence of the human SLAM2(pSURslam2), however, the position in the nucleotide sequence where thealternative exon is spliced is identical in both sequences (Table 2).

                                      TABLE 2                                     __________________________________________________________________________    Mouse SLAM sequences                                                          __________________________________________________________________________    Mouse SLAM1 (pMSLAM1) nucleotide and predicted amino acid                      sequence. Predicted leader sequences and the transmembrane                    sequence are underlined, though natural boundaries may be                     different, also depending upon cell type. Cysteines are found                 at amino acid residues numbered 32, 133, 161, 167, 212, 232,                  276, and 310. Potential N-linked glycosylation sites are                      found at residues numbered 54, 58, 103, 126, 151, 158, 192,                   210, and 226. Fragments between cysteines and/or N-linked                     glycosylation sites are particularly useful in generating                     antibodies.  SEQ ID NO: 9 AND 10.                                            __________________________________________________________________________            10        20        30        40        50        60                   tcctgccgagctgagctgagctgagctcacagctgggaccctgtctgcgattgctggcta                   -         70        80        90       100       110       120               atggatcccaaaggatccctttcctggagaatacttctgtttctctccctggcttttgag                   M  D  P  K  G  S  L  S  W  R  I  L  L  F  L  S  L  A  F  E                   1                                                                              -        130       140       150       160       170       180               ttgagctacggaacaggtggaggtgtgatggattgcccagtgattctccagaagctggga                   L  S  Y  G  T  G  G  G  V  M  D  C  P  V  I  L  Q  K  L  G                   21                                                                             -        190       200       210       220       230       240               caggacacgtggctgcccctgacgaatgaacatcagataaataagagcgtgaacaaaagt                   Q  D  T  W  L  P  L  T  N  E  H  Q  I  N  K  S  V  N  K  S                   41                                                                             -        250       260       270       280       290       300               gtccgcatcctcgtcaccatggcgacgtccccaggaagcaaatccaacaagaaaattgtg                   V  R  I  L  V  T  M  A  T  S  P  G  S  K  S  N  K  K  I  V                   61                                                                             -        310       320       330       340       350       360               tcttttgatctctctaaagggagctatccagatcacctggaggatggctaccactttcaa                   S  F  D  L  S  K  G  S  Y  P  D  H  L  E  D  G  Y  H  F  Q                   81                                                                             -        370       380       390       400       410       420               tcgaaaaacctgagcctgaagatcctcgggaacaggcgggagagtgaaggatggtacttg                   S  K  N  L  S  L  K  I  L  G  N  R  R  E  S  E  G  W  Y  L                   101                                                                            -        430       440       450       460       470       480               gtgagcgtggaggagaacgtttctgttcagcaattctgcaagcagctgaagctttatgaa                   V  S  V  E  E  N  V  S  V  Q  Q  F  C  K  Q  L  K  L  Y  E                   121                                                                            -        490       500       510       520       530       540               caggtctcccctccagagattaaagtgctaaacaaaacccaggagaacgagaatgggacc                   Q  V  S  P  P  E  I  K  V  L  N  K  T  Q  E  N  E  N  G  T                   141                                                                            -        550       560       570       580       590       600               tgcagcttgctgttggcctgcacagtgaagaaaggggaccatgtgacttacagctggagt                   C  S  L  L  L  A  C  T  V  K  K  G  D  H  V  T  Y  S  W  S                   161                                                                            -        610       620       630       640       650       660               gatgaggcaggcacccacctgctgagccgagccaaccgctcccacctcctgcacatcact                   D  E  A  G  T  H  L  L  S  R  A  N  R  S  H  L  L  H  I  T                   181                                                                            -        670       690       690       700       710       720               cttagcaaccagcatcaagacagcatctacaactgcaccgcaagcaaccctgtcagcagt                   L  S  N  Q  H  Q  D  S  I  Y  N  C  T  A  S  N  P  V  S  S                   201                                                                            -        730       740       750       760       770       780               atctctaggaccttcaacctatcatcgcaagcatgcaagcaggaatcctcctcagaatcg                   I  S  R  T  F  N  L  S  S  Q  A  C  K  Q  E  S  S  S  E  S                   221                                                                            -        790       800       810       820       830       840               agtccatggatgcaatatactcttgtaccactgggggtcgttataatcttcatcctggtt                   S  P  W  M  Q  Y  T  L  V  P  L  G  V  V  I  I  F  I  L  V                   241                                                                            -        850       860       870       880       890       900               ttcacggcaataataatgatgaaaagacaaggtaaatcaaatcactgccagccaccagtg                   F  T  A  I  I  M  M  K  R  Q  G  K  S  N  H  C  Q  P  P  V                   261                                                                            -        910       920       930       940       950       960               gaagaaaaaagccttactatttatgcccaagtacagaaatcagggcctcaagagaagaaa                   E  E  K  S  L  T  I  Y  A  Q  V  Q  K  S  G  P  Q  E  K  K                   281                                                                            -        970       980       990      1000      1010      1020               cttcatgatgccctaacagatcaggacccctgcacaaccatttatgtggctgccacagag                   L  H  D  A  L  T  D  Q  D  P  C  T  T  I  Y  V  A  A  T  E                   301                                                                            -       1030      1040      1050      1060      1070      1080               cctgccccagagtctgtccaggaaccaaaccccaccacagtttatgccagtgtgacactg                   P  A  P  E  S  V  Q  E  P  N  P  T  T  V  Y  A  S  V  T  L                   321                                                                            -       1090      1100      1110      1120      1130      1140               ccagagagctgacccatatacccagtgaaaggactttttgaaggaggatagaagaaccaa                   P  E  S                                                                      341                                                                            -       1150      1160      1170      1180      1190      1200               aatccacactgaactggaccccggggtccaagttctctgtgacagaaactgcacatctgt                 __________________________________________________________________________      -                                                                           Mouse SLAM2 (pMSLAM2) nucleotide and predicted amino acid                      sequence. The point after which the sequence of mouse SLAM2                   differ from mouse SLAM1 is indicated by • (nucleotide                   944).  SEQ ID NO: 11 and 12.                                                 __________________________________________________________________________            10        20        30        40        50        60                   tcctgccgagctgagctgagctgagctcacagctgggaccctgtctgcgattgctggcta                   -         70        80        90       100       110       120               atggatcccaaaggatccctttcctggagaatacttctgtttctctccctggcttttgag                   M  D  P  K  G  S  L  S  W  R  I  L  L  F  L  S  L  A  F  E                   1                                                                              -        130       140       150       160       170       180               ttgagctacggaacaggtggaggtgtgatggattgcccagtgattctccagaagctggga                   L  S  Y  G  T  G  G  G  V  M  D  C  P  V  I  L  Q  K  L  G                   21                                                                             -        190       200       210       220       230       240               caggacacgtggctgcccctgacgaatgaacatcagataaataagagcgtgaacaaaagt                   Q  D  T  W  L  P  L  T  N  E  H  Q  I  N  K  S  V  N  K  S                   41                                                                             -        250       260       270       280       290       300               gtccgcatcctcgtcaccatggcgacgtccccaggaagcaaatccaacaagaaaattgtg                   V  R  I  L  V  T  M  A  T  S  P  G  S  K  S  N  K  K  I  V                   61                                                                             -        310       320       330       340       350       360               tcttttgatctctctaaagggagctatccagatcacctggaggatggctaccactttcaa                   S  F  D  L  S  K  G  S  Y  P  D  H  L  E  D  G  Y  H  F  Q                   81                                                                             -        370       380       390       400       410       420               tcgaaaaacctgagcctgaagatcctcgggaacaggcgggagagtgaaggatggtacttg                   S  K  N  L  S  L  K  I  L  G  N  R  R  E  S  E  G  W  Y  L                   101                                                                            -        430       440       450       460       470       480               gtgagcgtggaggagaacgtttctgttcagcaattctgcaagcagctgaagctttatgaa                   V  S  V  E  E  N  V  S  V  Q  Q  F  C  K  Q  L  K  L  Y  E                   121                                                                            -        490       500       510       520       530       540               caggtctcccctccagagattaaagtgctaaacaaaacccaggagaacgagaatgggacc                   Q  V  S  P  P  E  I  K  V  L  N  K  T  Q  E  N  E  N  G  T                   141                                                                            -        550       560       570       580       590       600               tgcagcttgctgttggcctgcacagtgaagaaaggggaccatgtgacttacagctggagt                   C  S  L  L  L  A  C  T  V  K  K  G  D  H  V  T  Y  S  W  S                   161                                                                            -        610       620       630       640       650       660               gatgaggcaggcacccacctgctgagccgagccaaccgctcccacctcctgcacatcact                   D  E  A  G  T  H  L  L  S  R  A  N  R  S  H  L  L  H  I  T                   181                                                                            -        670       690       690       700       710       720               cttagcaaccagcatcaagacagcatctacaactgcaccgcaagcaaccctgtcagcagt                   L  S  N  Q  H  Q  D  S  I  Y  N  C  T  A  S  N  P  V  S  S                   201                                                                            -        730       740       750       760       770       780               atctctaggaccttcaacctatcatcgcaagcatgcaagcaggaatcctcctcagaatcg                   I  S  R  T  F  N  L  S  S  Q  A  C  K  Q  E  S  S  S  E  S                   221                                                                            -        790       800       810       820       830       840               agtccatggatgcaatatactcttgtaccactgggggtcgttataatcttcatcctggtt                   S  P  W  M  Q  Y  T  L  V  P  L  G  V  V  I  I  F  I  L  V                   241                                                                            -        850       860       870       880       890       900               ttcacggcaataataatgatgaaaagacaaggtaaatcaaatcactgccagccaccagtg                   F  T  A  I  I  M  M  K  R  Q  G  K  S  N  H  C  Q  P  P  V                   261                                                                            -        910       920       930       940       950       960               gaagaaaaaagccttactatttatgcccaagtacagaaatcaggggtacgttctatgcct                   E  E  K  S  L  T  I  Y  A  Q  V  Q  K  S  G  V  R  S  M  P                   281                                                                            -        970       980       990      1000      1010      1020               caccttgcgggagtgtctgtcatatttcgcacaggatttctgatagctgccttgcacaca                   H  L  A  G  V  S  V  I  F  R  T  G  F  L  I  A  A  L  H  T                   301                                                                            -       1030      1040      1050      1060      1070      1080               accatggtcctgcagggactcctagagtagatgaacttaagaaagcagaaaagtcaagaa                   T  M  V  L  Q  G  L  L  E                                                    321                                                                            -       1090      1100      1110      1120      1130      1140               caagagctcccccagtgtcactgacccttatattgtttgaacttgtagaaaacagtgaca                 __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    Alignment of mouse SLAM1 to human SLAM1.                                      __________________________________________________________________________                                        •                                         M   1' MDPKGSLSWRILLFLSLAFELSYGTGGGVMDCPVILQKLGQDTWLPLTNEHQINKSVNKS        ***** **  ..*******. ****** .*.** **..** .. **** *. ****.***                 H   1" MDPKGLLSLTFVLFLSLAFGASYGTGGRMMNCPKILRQLGSKVLLPLTYER-INKSMNKS                - M  61' VRILVTMATSPGSKSNKKIVSFDLSKGSYPDHLEDGYHFQSKNLSLKILGNRRESEGW         YL                                                                          ..*.****.* ... ..****.* *... * .*.* *.*  .**.* * ..*.*.*****                 H  60" IHIVVTMAKSLENSVENKIVSLDPSEAGPPRYLGDRYKFYLENLTLGIRESRKEDEGWYL                           •                            •     •            M 121' VSVEENVSVQQFCKQLKLYEQVSPPEIKVLNKTQENENGTCSLLLACTVKKGDHVTYSWS        ...*.*****.** **.******.**********  *****.*.*.***.*****.****                 H 120" MTLEKNVSVQRFCLQLRLYEQVSTPEIKVLNKTQ--ENGTCTLILGCTVEKGDHVAYSWS                                               •                                   M 181' DEAGTHLLSRANRSHLLHITLSNQHQDSIYNCTASNPVSSISRTFNLSSQACKQESSSES                ..**** *..**.**** .**. **.*.** **.***.*. *.**. . ..*. . .**.                 H 178" EKAGTHPLNPANSSHLLSLTLGPQHADNIYICTVSNPISNNSQTFS-PWPGCRTD-PSET        - M 241' SPWMQYTLVPLGVVIIFILVFTAIIMMKRQGKSNHCQPPVEEKSLTIYAQVQKSGPQEKK             .**  *. . ** **. **....*. ..*.**.**.*..**.***********.** .**                 H 236" KPWAVYAGL-LGGVIM-ILIMVVILQLRRRGKTNHYQTTVEKKSLTIYAQVQKPGPLQKK                  •                                                            M 301' LHDALTDQDPCTTIYVAATEPAPESVQEPNPTTVYASVTLPES                             * *....**************.******.*..********                                     H 294" L-DSFPAQDPCTTIYVAATEPVPESVQETNSITVYASVTLQRADTRDQQRDFLKENGKTK         __________________________________________________________________________     • indicates a conserved cysteine;                                       * indicates identical amino acids;                                            . indicates a conserved amino acid;                                             conserved cysteines in the cytoplasmic domain are underlined.          

Some homology is apparent in the extracellular domains of human SLAMwith mouse 2B4, human CD48, and human LFA-3 (CD58) protein sequences.Alignment of the sequences reveals portions of shared homology,disparate homology, common motifs, and partly shared features.

The natural antigens are capable of mediating various biochemicalresponses which lead to biological or physiological responses in targetcells. The best characterized embodiment was initially described inhuman, but human and mouse variants are also described herein.Additional sequences for proteins in other mammalian species, e.g.,primates and rodents, should also be available. See below. Thedescriptions below are directed, for exemplary purposes, to a humanSLAM, but are likewise applicable to related embodiments from otherspecies.

Isolated human SLAM protein is a protein which exhibits structuralfeatures characteristic of a cell surface antigen. The protein is easilydetected on particular cell types, others express lesser amounts. SeeTable 4. The SLAM mediates a biochemical response to binding of anantibody, or other yet unidentified ligands, leading to signaltransduction and cellular response. In particular, the SLAM antigen hasbeen isolated by expression cloning using a specific antibody. The SLAMantigen was isolated and characterized as a protein which migrates onpolyacrylamide gel electrophoresis with a mobility characteristic of aprotein of about 70 kD. The core protein, after treatment withN-glycanase, has a mobility of about a 40 kd protein.

                  TABLE 4                                                         ______________________________________                                        Cellular expression of SLAM. RNA from various cells and tissues                 was subject to reverse transcription and PCR using SLAM specific             primers. Rough qualitative determinations are provided, though a              negative merely means below threshold detection levels. Thymus also           expresses the message.                                                             cell type         expression                                            ______________________________________                                        JY EBV transformed B cells                                                                        +                                                           purified B cells CD20.sup.+ +                                                 CD4.sup.+  T-cell clone S11 +                                                 CD4.sup.+  T-cell clone S40 +                                                 CD4.sup.+  T-cell clone B21 +                                                 CD4.sup.+  T-cell clone B21 activated +                                       purified NK cells +                                                           purified NK cells +                                                           fetal liver -                                                                 fetal bone marrow -                                                           fetal thymus +                                                                small intestine -                                                             brain -                                                                       kidney -                                                                      heart -                                                                       FL508 pre-T cell line +                                                       TN92 pre-T cell line +                                                      ______________________________________                                    

The SLAM antigen should be present in the identified tissue types andthe interaction of the antigen with its binding partner should beimportant for mediating various aspects of cellular physiology ordevelopment.

II. Purified SLAM

Human and mouse SLAM amino acid sequences are shown in SEQ ID NO: 2, 4,6, 8, 10, and 12. These amino acid sequences, provided amino to carboxy,are important in providing sequence information in the antigen allowingfor distinguishing the protein from other proteins and exemplifyingnumerous variants. Moreover, the peptide sequences allow preparation ofpeptides to generate antibodies to recognize such segments, and allowpreparation of oligonucleotide probes, both of which are strategies fordetection or isolation, e.g., cloning, of genes encoding such sequences.

As used herein, the term "human SLAM" shall encompass, when used in aprotein context, a protein having amino acid sequences shown in SEQ IDNO: 2, 4, 6, or 8, or a significant fragment of such a protein, oranother highly homologous protein derived from human. Clearly, there aremRNA species representing splicing variants. It also refers to a humanderived polypeptide which exhibits similar biological function orinteracts with SLAM specific binding components. These bindingcomponents, e.g., antibodies, typically bind to a SLAM with highaffinity, e.g., at least about 100 nM, usually better than about 30 nM,preferably better than about 10 nM, and more preferably at better thanabout 3 nM. Homologous proteins would be found in mammalian speciesother than human, e.g., primates or rodents. Non-mammalian speciesshould also possess structurally or functionally related genes andproteins, e.g., birds or amphibians.

The term "polypeptide" as used herein includes a significant fragment orsegment, and encompasses a stretch of amino acid residues of at leastabout 8 amino acids, generally at least about 12 amino acids, typicallyat least about 16 amino acids, preferably at least about 20 amino acids,and, in particularly preferred embodiments, at least about 30 or moreamino acids.

The term "binding composition" refers to molecules that bind withspecificity to SLAM, e.g., in a cell adhesion pairing type fashion, oran antibody-antigen interaction. It also includes compounds, e.g.,proteins, which specifically associate with SLAM, including in a naturalphysiologically relevant protein-protein interaction, either covalent ornon-covalent. The molecule may be a polymer, or chemical reagent. Afunctional analog may be an antigen with structural modifications, or itmay be a molecule which has a molecular shape which interacts with theappropriate binding determinants. The compounds may serve as agonists orantagonists of the binding interaction, see, e.g., Goodman, et al.(eds.) (1990) Goodman & Gilman's: The Pharmacological Bases ofTherapeutics (8th ed.), Pergamon Press.

Substantially pure typically means that the protein is free from othercontaminating proteins, nucleic acids, or other biologicals derived fromthe original source organism. Purity may be assayed by standard methods,typically by weight, and will ordinarily be at least about 40% pure,generally at least about 50% pure, often at least about 60% pure,typically at least about 80% pure, preferably at least about 90% pure,and in most preferred embodiments, at least about 95% pure. Carriers orexcipients will often be added.

Solubility of a polypeptide or fragment depends upon the environment andthe polypeptide. Many parameters affect polypeptide solubility,including temperature, electrolyte environment, size and molecularcharacteristics of the polypeptide, and nature of the solvent.Typically, the temperature at which the polypeptide is used ranges fromabout 4° C. to about 65° C. Usually the temperature at use is greaterthan about 18° C. For diagnostic purposes, the temperature will usuallybe about room temperature or warmer, but less than the denaturationtemperature of components in the assay. For therapeutic purposes, thetemperature will usually be body temperature, typically about 37° C. forhumans and mice, though under certain situations the temperature may beraised or lowered in situ or in vitro.

The size and structure of the polypeptide should generally be in asubstantially stable state, and usually not in a denatured state. Thepolypeptide may be associated with other polypeptides in a quaternarystructure, e.g., to confer solubility, or associated with lipids ordetergents in a manner which approximates natural lipid bilayerinteractions.

The solvent and electrolytes will usually be a biologically compatiblebuffer, of a type used for preservation of biological activities, andwill usually approximate a physiological aqueous solvent. Usually thesolvent will have a neutral pH, typically between about 5 and 10, andpreferably about 7.5. On some occasions, one or more detergents will beadded, typically a mild non-denaturing one, e.g., CHS (cholesterylhemisuccinate) or CHAPS (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), or a low enough concentration as to avoid significantdisruption of structural or physiological properties of the protein.

III. Physical Variants

This invention also encompasses proteins or peptides having substantialamino acid sequence identity with the amino acid sequence of the SLAM.The variants include species or allelic variants.

Amino acid sequence homology, or sequence identity, is determined byoptimizing residue matches, if necessary, by introducing gaps asrequired. See also Needleham, et al. (1970) J. Mol. Biol. 48:443-453;Sankoff, et al. (1983) Chapter One in Time Warps, Strina Edits, andMacromolecules: The Theory and Practice of Seauence Comparison,Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif.; and the University of WisconsinGenetics Computer Group, Madison, Wis. Sequence identity changes whenconsidering conservative substitutions as matches. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine. Homologous amino acid sequencesare typically intended to include natural allelic and interspeciesvariations in each respective protein sequence. Typical homologousproteins or peptides will have from 25-100% identity (if gaps can beintroduced), to 50-100% identity (if conservative substitutions areincluded) with the amino acid sequence of the SLAM. Identity measureswill be at least about 35%, generally at least about 40%, often at leastabout 50%, typically at least about 60%, usually at least about 70%,preferably at least about 80%, and more preferably at least about 90%.

The isolated SLAM DNA can be readily modified by nucleotidesubstitutions, nucleotide deletions, nucleotide insertions, andinversions of nucleotide stretches. These modifications result in novelDNA sequences which encode these antigens, their derivatives, orproteins having similar physiological, immunogenic, antigenic, or otherfunctional activity. These modified sequences can be used to producemutant antigens or to enhance expression. Enhanced expression mayinvolve gene amplification, increased transcription, increasedtranslation, and other mechanisms. "Mutant SLAM" encompasses apolypeptide otherwise falling within the sequence identity definition ofthe SLAM as set forth above, but having an amino acid sequence whichdiffers from that of SLAM as normally found in nature, whether by way ofdeletion, substitution, or insertion. This generally includes proteinshaving significant identity with a protein having sequences of SEQ IDNO: 2, 4, 6, 8, 10, or 12, and as sharing various biological activities,e.g., antigenic or immunogenic, with those sequences, and in preferredembodiments contain most of the full length disclosed sequences. Fulllength sequences will typically be preferred, though truncated versionswill also be useful. Similar concepts apply to different SLAM proteins,particularly those found in various warm blooded animals, e.g., mammalsand birds. These descriptions are generally meant to encompass all SLAMproteins, not limited to the particular human or mouse embodimentsspecifically discussed.

SLAM mutagenesis can also be conducted by making amino acid insertionsor deletions. Substitutions, deletions, insertions, or any combinationsmay be generated to arrive at a final construct. Insertions includeamino- or carboxy- terminal fusions. Random mutagenesis can be conductedat a target codon and the expressed mutants can then be screened for thedesired activity. Methods for making substitution mutations atpredetermined sites in DNA having a known sequence are well known in theart, e.g., by M13 primer mutagenesis or polymerase chain reaction (PCR)techniques. See, e.g., Sambrook, et al. (1989); Ausubel, et al. (1987and Supplements); and Kunkel, et al. (1987) Methods in Enzymol.154:367-382.

The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these proteins. Aheterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. A similar conceptapplies to heterologous nucleic acid sequences.

In addition, new constructs may be made from combining similarfunctional domains from other proteins. For example, target-binding orother segments may be "swapped" between different new fusionpolypeptides or fragments. See, e.g., Cunningham, et al. (1989) Science243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence, e.g., PCRtechniques.

IV. Functional Variants

The blocking of physiological response to SLAMs may result from theinhibition of binding of the antigen to its binding partner, e.g.,another of itself, likely through competitive inhibition. Thus, in vitroassays of the present invention will often use isolated protein,membranes from cells expressing a membrane associated recombinant SLAM,soluble fragments comprising antigen binding segments of these proteins,or fragments attached to solid phase substrates. These assays will alsoallow for the diagnostic determination of the effects of either bindingsegment mutations and modifications, or antigen mutations andmodifications, e.g., SLAM analogs.

This invention also contemplates the use of competitive drug screeningassays, e.g., where neutralizing antibodies to antigen or bindingfragments compete with a test compound for binding to the protein."Derivatives" of SLAM antigens include amino acid sequence mutants,glycosylation variants, and covalent or aggregate conjugates with otherchemical moieties. Covalent derivatives can be prepared by linkage offunctionalities to groups which are found in SLAM amino acid side chainsor at the N- or C-termini, e.g., by standard means. See, e.g., Lundbladand Noyes (1988) Chemical Reagents for Protein Modification, vols. 1-2,CRC Press, Inc., Boca Raton, Fla.; Hugli (ed.) (1989) Techniques inProtein Chemistry, Academic Press, San Diego, Calif.; and Wong (1991)Chemistry of Protein Conjugation and Cross Linking, CRC Press, BocaRaton, Fla.

In particular, glycosylation alterations are included, e.g., made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. See, e.g.,Elbein (1987) Ann. Rev. Biochem. 56:497-534. Also embraced are versionsof the peptides with the same primary amino acid sequence which haveother minor modifications, including phosphorylated amino acid residues,e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

Fusion polypeptides between SLAMs and other homologous or heterologousproteins are also provided. Many cytokine receptors or other surfaceproteins are multimeric, e.g., homodimeric entities, and a repeatconstruct may have various advantages, including lessened susceptibilityto proteolytic cleavage. Typical examples are fusions of a reporterpolypeptide, e.g., luciferase, with a segment or domain of a protein,e.g., a receptor-binding segment, so that the presence or location ofthe fused ligand may be easily determined. See, e.g., Dull, et al., U.S.Pat. No. 4,859,609. Other gene fusion partners include bacterialβ-galactosidase, trpE, Protein A, β-lactamase, alpha amylase, alcoholdehydrogenase, yeast alpha mating factor, and detection or purificationtags such as a FLAG sequence or His6 sequence. See, e.g., Godowski, etal. (1988) Science 241:812-816.

Fusion peptides will typically be made by either recombinant nucleicacid methods or by synthetic polypeptide methods. Techniques for nucleicacid manipulation and expression are described generally, e.g., inSambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.),vols. 1-3, Cold Spring Harbor Laboratory; and Ausubel, et al. (eds.)(1993) Current Protocols in Molecular Biology, Greene and Wiley, NY.Techniques for synthesis of polypeptides are described, e.g., inMerrifield (1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986)Science 232: 341-347; Atherton, et al. (1989) Solid Phase PeptideSynthesis: A Practical Aporoach, IRL Press, Oxford; and Grant (1992)Synthetic Peptides: A User's Guide, W. H. Freeman, NY.

This invention also contemplates the use of derivatives of SLAMs otherthan variations in amino acid sequence or glycosylation. Suchderivatives may involve covalent or aggregative association withchemical moieties. Covalent or aggregative derivatives will be useful asimmunogens, as reagents in immunoassays, or in purification methods suchas for affinity purification of binding partners, e.g., other antigens.A SLAM can be immobilized by covalent bonding to a solid support such ascyanogen bromide-activated SEPHAROSE, by methods which are well known inthe art, or adsorbed onto polyolefin surfaces, with or withoutglutaraldehyde cross-linking, for use in the assay or purification ofanti-SLAM antibodies or an alternative binding composition. The SLAMscan also be labeled with a detectable group, e.g., for use in diagnosticassays. Purification of SLAM may be effected by an immobilized antibodyor complementary binding partner.

A solubilized SLAM or fragment of this invention can be used as animmunogen for the production of antisera or antibodies specific forbinding to the antigen or fragments thereof. Purified antigen can beused to screen monoclonal antibodies or antigen-binding fragments,encompassing antigen binding fragments of natural antibodies. PurifiedSLAMs can also be used as a reagent to detect antibodies generated inresponse to the presence of elevated levels of the antigen or cellfragments containing the antigen, both of which may be diagnostic of anabnormal or specific physiological or disease condition. This inventioncontemplates antibodies raised against amino acid sequences encoded bynucleotide sequences shown in SEQ ID NO: 1, 3, 5, 7, 9, or 11, orfragments of proteins containing them. In particular, this inventioncontemplates antibodies having binding affinity to or being raisedagainst specific fragments which are predicted to lie outside of thelipid bilayer, both extracellular or intracellular.

The present invention contemplates the isolation of additional closelyrelated species variants. Southern and Northern blot analysis shouldestablish that similar genetic entities exist in other mammals. It islikely that SLAMs are widespread in species variants, e.g., rodents,lagomorphs, carnivores, artiodactyla, perissodactyla, and primates.

The invention also provides means to isolate a group of related antigensdisplaying both distinctness and similarities in structure, expression,and function. Elucidation of many of the physiological effects of themolecules will be greatly accelerated by the isolation andcharacterization of additional distinct species variants of them. Inparticular, the present invention provides useful probes for identifyingadditional homologous genetic entities in different species.

The isolated genes will allow transformation of cells lacking expressionof a corresponding SLAM, e.g., either species types or cells which lackcorresponding antigens and exhibit negative background activity. Thisshould allow analysis of the function of SLAM in comparison tountransformed control cells.

Dissection of critical structural elements which effect the variousactivation or differentiation functions mediated through these antigensis possible using standard techniques of modern molecular biology,particularly in comparing members of the related class. See, e.g., thehomolog-scanning mutagenesis technique described in Cunningham, et al.(1989) Science 243:1339-1336; and approaches used in O'Dowd, et al.(1988) J. Biol. Chem. 263:15985-15992; and Lechleiter, et al. (1990)EMBO J. 9:4381-4390.

Intracellular functions would probably involve segments of the antigenwhich are normally accessible to the cytosol. However, proteininternalization may occur under certain circumstances, and interactionbetween intracellular components and "extracellular" segments may occur.The specific segments of interaction of SLAM with other intracellularcomponents may be identified by mutagenesis or direct biochemical means,e.g., cross-linking or affinity methods. Structural analysis bycrystallographic or other physical methods will also be applicable.Further investigation of the mechanism of signal transduction willinclude study of associated components which may be isolatable byaffinity methods or by genetic means, e.g., complementation analysis ofmutants.

Further study of the expression and control of SLAM will be pursued. Thecontrolling elements associated with the antigens should exhibitdifferential physiological, developmental, tissue specific, or otherexpression patterns. Upstream or downstream genetic regions, e.g.,control elements, are of interest. In particular, physiological ordevelopmental variants, e.g., multiple alternatively processed forms ofthe antigen have been found. See, e.g., SEQ ID NO: 1 and 3. Thus,differential splicing of message may lead to an assortment of membranebound forms, soluble forms, and modified versions of antigen.

Structural studies of the antigens will lead to design of new antigens,particularly analogs exhibiting agonist or antagonist properties on themolecule. This can be combined with previously described screeningmethods to isolate antigens exhibiting desired spectra of activities.

V. Antibodies

Antibodies can be raised to various SLAMs, including species or allelicvariants, and fragments thereof, both in their naturally occurring formsand in their recombinant forms. Additionally, antibodies can be raisedto SLAMs in either their active forms or in their inactive forms,including native or denatured versions. Anti-idiotypic antibodies arealso contemplated.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of the antigens can be raised byimmunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective SLAMs, or screened for agonistic orantagonistic activity, e.g., mediated through the antigen or its bindingpartner. These monoclonal antibodies will usually bind with at least aK_(D) of about 1 mM, more usually at least about 300 μM, typically atleast about 100 μM, more typically at least about 30 μM, preferably atleast about 10 μM, and more preferably at least about 3 μM or better.

The antibodies of this invention can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they can bescreened for ability to bind to the antigens without inhibiting bindingby a partner. As neutralizing antibodies, they can be useful incompetitive binding assays. They will also be useful in detecting orquantifying SLAM protein or its binding partners. See, e.g., Chan (ed.)(1987) Immunology: A Practical Guide, Academic Press, Orlando, Fla.;Price and Newman (eds.) (1991) Principles and Practice of Immunoassay,Stockton Press, N.Y.; and Ngo (ed.) (1988) Nonisotopic Immunoassay,Plenum Press, N.Y. Cross absorptions or other tests will identifyantibodies which exhibit various spectra of specificities, e.g., uniqueor shared species specificities.

Further, the antibodies, including antigen binding fragments, of thisinvention can be potent antagonists that bind to the antigen and inhibitfunctional binding or inhibit the ability of a binding partner to elicita biological response. They also can be useful as non-neutralizingantibodies and can be coupled to toxins or radionuclides so that whenthe antibody binds to antigen, a cell expressing it, e.g., on itssurface, is killed. Further, these antibodies can be conjugated to drugsor other therapeutic agents, either directly or indirectly by means of alinker, and may effect drug targeting.

Antigen fragments may be joined to other materials, particularlypolypeptides, as fused or covalently joined polypeptides to be used asimmunogens. An antigen and its fragments may be fused or covalentlylinked to a variety of immunogens, such as keyhole limpet hemocyanin,bovine serum albumin, tetanus toxoid, etc. See Microbiology, HoeberMedical Division, Harper and Row, 1969; Landsteiner (1962) Specificityof Serological Reactions, Dover Publications, New York; Williams, et al.(1967) Methods in Immunology and Immunochemistry, vol. 1, AcademicPress, New York; and Harlow and Lane (1988) Antibodies: A LaboratoryManual, CSH Press, NY, for descriptions of methods of preparingpolyclonal antisera.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology(4th ed.), Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual,CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.), Academic Press, New York; and particularly in Kohler andMilstein (1975) in Nature 256:495-497, which discusses one method ofgenerating monoclonal antibodies.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides or alternatively to selection of libraries ofantibodies in phage or similar vectors. See, Huse, et al. (1989)"Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda," Science 246:1275-1281; and Ward, et al.(1989) Nature 341:544-546. The polypeptides and antibodies of thepresent invention may be used with or without modification, includingchimeric or humanized antibodies. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced, see Cabilly, U.S. Pat. No. 4,816,567; Moore, et al.,U.S. Pat. No. 4,642,334; and Queen, et al. (1989) Proc. Nat'l Acad. Sci.USA 86:10029-10033.

The antibodies of this invention can also be used for affinitychromatography in isolating the protein. Columns can be prepared wherethe antibodies are linked to a solid support. See, e.g., Wilchek et al.(1984) Meth. Enzymol. 104:3-55.

Antibodies raised against each SLAM will also be useful to raiseanti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of therespective antigens.

VI. Nucleic Acids

The described peptide sequences and the related reagents are useful indetecting, isolating, or identifying a DNA clone encoding SLAM, e.g.,from a natural source. Typically, it will be useful in isolating a genefrom mammal, and similar procedures will be applied to isolate genesfrom other species, e.g., warm blooded animals, such as birds andmammals. Cross hybridization will allow isolation of SLAM from otherspecies. A number of different approaches should be available tosuccessfully isolate a suitable nucleic acid clone.

The purified protein or defined peptides are useful for generatingantibodies by standard methods, as described above. Synthetic peptidesor purified protein can be presented to an immune system to generatemonoclonal or polyclonal antibodies. See, e.g., Coligan (1991) CurrentProtocols in Immunology Wiley/Greene; and Harlow and Lane (1989)Antibodies: A Laboratory Manual, Cold Spring Harbor Press.Alternatively, the SLAM can be used as a specific binding reagent, andadvantage can be taken of its specificity of binding, much like anantibody would be used.

For example, the specific binding composition could be used forscreening of an expression library made from a cell line which expressesa SLAM. The screening can be standard staining of surface expressedantigen, or by panning. Screening of intracellular expression can alsobe performed by various staining or immunofluorescence procedures. Thebinding compositions could be used to affinity purify or sort out cellsexpressing the protein.

The peptide segments can also be used to predict appropriateoligonucleotides to screen a library. The genetic code can be used toselect appropriate oligonucleotides useful as probes for screening. See,e.g., SEQ ID NO: 1 or 3. In combination with polymerase chain reaction(PCR) techniques, synthetic oligonucleotides will be useful in selectingcorrect clones from a library. Complementary sequences will also be usedas probes, primers, or antisense strands. Based upon identification ofthe likely extracellular domain, various fragments should beparticularly useful, e.g., coupled with anchored vector or poly-Acomplementary PCR techniques or with complementary DNA of otherpeptides.

This invention contemplates use of isolated DNA or fragments to encode abiologically active corresponding SLAM polypeptide. In addition, thisinvention covers isolated or recombinant DNA which encodes abiologically active protein or polypeptide which is capable ofhybridizing under appropriate conditions with the DNA sequencesdescribed herein. Said biologically active protein or polypeptide can bean intact antigen, or fragment, and have an amino acid sequencedisclosed in, e.g., SEQ ID NO: 1 or 3. Further, this invention coversthe use of isolated or recombinant DNA, or fragments thereof, whichencode proteins which are homologous to a SLAM or which was isolatedusing cDNA encoding a SLAM as a probe. The isolated DNA can have therespective regulatory sequences in the 5' and 3' flanks, e.g.,promoters, enhancers, poly-A addition signals, and others.

An "isolated" nucleic acid is a nucleic acid, e.g., an RNA, DNA, or amixed polymer, which is substantially separated from other componentswhich naturally accompany a native sequence, e.g., ribosomes,polymerases, and/or flanking genomic sequences from the originatingspecies. The term embraces a nucleic acid sequence which has beenremoved from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule.Generally, the nucleic acid will be in a vector or fragment less thanabout 50 kb, usually less than about 30 kb, typically less than about 10kb, and preferably less than about 6 kb.

An isolated nucleic acid will generally be a homogeneous composition ofmolecules, but will, in some embodiments, contain minor heterogeneity.This heterogeneity is typically found at the polymer ends or portionsnot critical to a desired biological function or activity.

A "recombinant" nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence, typically selection or production. Alternatively,it can be a nucleic acid made by generating a sequence comprising fusionof two fragments which are not naturally contiguous to each other, butis meant to exclude products of nature, e.g., naturally occurringmutants. Thus, e.g., products made by transforming cells with anyunnaturally occurring vector is encompassed, as are nucleic acidscomprising sequence derived using any synthetic oligonucleotide process.Such is often done to replace a codon with a redundant codon encodingthe same or a conservative amino acid, while typically introducing orremoving a sequence recognition site.

Alternatively, it is performed to join together nucleic acid segments ofdesired functions to generate a single genetic entity comprising adesired combination of functions not found in the commonly availablenatural forms. Restriction enzyme recognition sites are often the targetof such artificial manipulations, but other site specific targets, e.g.,promoters, DNA replication sites, regulation sequences, controlsequences, or other useful features may be incorporated by design. Asimilar concept is intended for a recombinant, e.g., fusion,polypeptide. Specifically included are synthetic nucleic acids which, bygenetic code redundancy, encode polypeptides similar to fragments ofthese antigens, and fusions of sequences from various different speciesvariants.

A significant "fragment" in a nucleic acid context is a contiguoussegment of at least about 17 nucleotides, generally at least about 22nucleotides, ordinarily at least about 29 nucleotides, more often atleast about 35 nucleotides, typically at least about 41 nucleotides,usually at least about 47 nucleotides, preferably at least about 55nucleotides, and in particularly preferred embodiments will be at leastabout 60 or more nucleotides.

A DNA which codes for a SLAM protein will be particularly useful toidentify genes, mRNA, and cDNA species which code for related orhomologous proteins, as well as DNAs which code for homologous proteinsfrom different species. There are likely homologues in other species,including primates, rodents, and birds. Various SLAM proteins should behomologous and are encompassed herein. However, even proteins that havea more distant evolutionary relationship to the antigen can readily beisolated under appropriate conditions using these sequences if they aresufficiently homologous. Primate SLAM proteins are of particularinterest.

Recombinant clones derived from the genomic sequences, e.g., containingintrons, will be useful for transgenic studies, including, e.g.,transgenic cells and organisms, and for gene therapy. See, e.g., Goodnow(1992) "Transgenic Animals" in Roitt (ed.) Encyclopedia of Immunology,Academic Press, San Diego, pp. 1502-1504; Travis (1992) Science256:1392-1394; Kuhn, et al. (1991) Science 254:707-710; Capecchi (1989)Science 244:1288; Robertson (1987)(ed.) Teratocarcinomas and EmbryonicStem Cells: A Practical Approach, IRL Press, Oxford; and Rosenberg(1992) J. Clinical Oncology 10:180-199.

Substantial homology in the nucleic acid sequence comparison contextmeans either that the segments, or their complementary strands, whencompared, are identical when optimally aligned, with appropriatenucleotide insertions or deletions, in at least about 50% of thenucleotides, generally at least about 58%, ordinarily at least about65%, often at least about 71%, typically at least about 77%, usually atleast about 85%, preferably at least about 95 to 98% or more, and inparticular embodiments, as high as about 99% or more of the nucleotides.Alternatively, substantial homology exists when the segments willhybridize under selective hybridization conditions, to a strand, or itscomplement, typically using a sequence of SLAM, e.g., in SEQ ID NO: 1,3, 5, 7, 9, or 11. Typically, selective hybridization will occur whenthere is at least about 55% homology over a stretch of at least about 30nucleotides, preferably at least about 75% over a stretch of about 25nucleotides, and most preferably at least about 90% over about 20nucleotides. See, Kanehisa (1984) Nuc. Acids Res. 12:203-213. The lengthof homology comparison, as described, may be over longer stretches, andin certain embodiments will be over a stretch of at least about 17nucleotides, usually at least about 28 nucleotides, typically at leastabout 40 nucleotides, and preferably at least about 75 to 100 or morenucleotides.

Stringent conditions, in referring to homology in the hybridizationcontext, will be stringent combined conditions of salt, temperature,organic solvents, and other parameters, typically those controlled inhybridization reactions. Stringent temperature conditions will usuallyinclude temperatures in excess of about 30° C., usually in excess ofabout 37° C., typically in excess of about 55° C., preferably in excessof about 70° C. Stringent salt conditions will ordinarily be less thanabout 1000 mM, usually less than about 400 mM, typically less than about250 mM, preferably less than about 150 mM. However, the combination ofparameters is much more important than the measure of any singleparameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol.31:349-370.

SLAM from other mammalian species can be cloned and isolated bycross-species hybridization of closely related species. Homology may berelatively low between distantly related species, and thus hybridizationof relatively closely related species is advisable. Alternatively,preparation of an antibody preparation which exhibits less speciesspecificity may be useful in expression cloning approaches.

VII. Making SLAM; Mimetics

DNA which encodes the SLAM or fragments thereof can be obtained bychemical synthesis, screening cDNA libraries, or screening genomiclibraries prepared from a wide variety of cell lines or tissue samples.See, e.g., Okayama and Berg (1982) Mol. Cell. Biol. 2:161-170; Gublerand Hoffman (1983) Gene 25:263-269; and Glover (ed.) (1984) DNA Cloning:A Practical Approach, IRL Press, Oxford. Alternatively, the sequencesprovided herein provide useful PCR primers or allow synthetic or otherpreparation of suitable genes encoding a SLAM.

This DNA can be expressed in a wide variety of host cells for thesynthesis of a full-length SLAM or fragments which can in turn, e.g., beused to generate polyclonal or monoclonal antibodies; for bindingstudies; for construction and expression of modified molecules; and forstructure/function studies.

Vectors, as used herein, comprise plasmids, viruses, bacteriophage,integratable DNA fragments, and other vehicles which enable theintegration of DNA fragments into the genome of the host. See, e.g.,Pouwels, et al. (1985 and Supplements) Clonina Vectors: A LaboratoryManual, Elsevier, N.Y.; and Rodriguez, et al. (1988)(eds.) Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, Buttersworth,Boston, Mass.

For purposes of this invention, DNA sequences are operably linked whenthey are functionally related to each other. For example, DNA for apresequence or secretory leader is operably linked to a polypeptide ifit is expressed as a preprotein or participates in directing thepolypeptide to the cell membrane or in secretion of the polypeptide. Apromoter is operably linked to a coding sequence if it controls thetranscription of the polypeptide; a ribosome binding site is operablylinked to a coding sequence if it is positioned to permit translation.Usually, operably linked means contiguous and in reading frame, however,certain genetic elements such as repressor genes are not contiguouslylinked but still bind to operator sequences that in turn controlexpression. See e.g., Rodriguez, et al., Chapter 10, pp. 205-236; Balbasand Bolivar (1990) Methods in Enzymology 185:14-37; and Ausubel, et al.(1993) Current Protocols in Molecular Biology, Greene and Wiley, NY.

Representative examples of suitable expression vectors include pCDNA1;pCD, see Okayama, et al. (1985) Mol. Cell Biol. 5:1136-1142; pMClneoPoly-A, see Thomas, et al. (1987) Cell 51:503-512; and a baculovirusvector such as pAC 373 or pAC 610. See, e.g., Miller (1988) Ann. Rev.Microbiol. 42:177-199.

It will often be desired to express a SLAM polypeptide in a system whichprovides a specific or defined glycosylation pattern. See, e.g., Luckowand Summers (1988) Bio/Technology 6:47-55; and Kaufman (1990) Meth.Enzymol. 185:487-511.

The SLAM, or a fragment thereof, may be engineered to be phosphatidylinositol (PI) linked to a cell membrane, but can be removed frommembranes by treatment with a phosphatidyl inositol cleaving enzyme,e.g., phosphatidyl inositol phospholipase-C. This releases the antigenin a biologically active form, and allows purification by standardprocedures of protein chemistry. See, e.g., Low (1989) Biochim. Biophys.Acta 988:427-454; Tse, et al. (1985) Science 230:1003-1008; and Brunner,et al. (1991) J. Cell Biol. 114:1275-1283.

Now that the SLAM has been characterized, fragments or derivativesthereof can be prepared by conventional processes for synthesizingpeptides. These include processes such as are described in Stewart andYoung (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co.,Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice of PeptideSynthesis, Springer-Verlag, New York; Bodanszky (1984) The Principles ofPeptide Synthesis, Springer-Verlag, New York; and Villafranca (ed.)(1991) Techniques in Protein Chemistry II, Academic Press, San Diego,Calif.

VIII. Uses

The present invention provides reagents which will find use indiagnostic applications as described elsewhere herein, e.g., in thegeneral description for T cell mediated conditions, or below in thedescription of kits for diagnosis.

This invention also provides reagents with significant therapeuticvalue. The SLAM (naturally occurring or recombinant), fragments thereof,and antibodies thereto, along with compounds identified as havingbinding affinity to SLAM, should be useful in the treatment ofconditions associated with abnormal physiology or development, includingabnormal proliferation, e.g., cancerous conditions, or degenerativeconditions. In particular, modulation of development of lymphoid cellswill be achieved by appropriate therapeutic treatment using thecompositions provided herein. For example, a disease or disorderassociated with abnormal expression or abnormal signaling by a SLAMshould be a likely target for an agonist or antagonist of the antigen.The antigen plays a role in regulation or development of hematopoieticcells, e.g., lymphoid cells, which affect immunological responses, e.g.,autoimmune disorders.

In particular, the antigen has been demonstrated to provide acostimulatory signal to T cell activation. Thus, the SLAM has a role inT cell to T cell interactions. These interactions lead, in particularcontexts, to cell proliferation, enhanced cytokine synthesis by thecells, and consequential amplification of T cell proliferation.

Moreover, the SLAM induced production of interferon-γ suggests thatcertain agonists to SLAM could direct T cell responses towards a Th0/Th1pathway, and thus suppress a Th2 type response. Among these agonistsshould be various antibodies which recognize the appropriate epitopes,e.g., which mimic binding of SLAM to its ligand.

Conversely, antagonists of SLAM, such as the naturally occurringsecreted form of SLAM or blocking antibodies, may provide a selectiveand powerful way to block immune responses in abnormal situations, e.g.,autoimmune disorders, including rheumatoid arthritis, systemic lupuserythematosis (SLE), Hashimoto's autoimmune thyroiditis, as well asacute and chronic inflammatory responses in which T cell activation,expansion, and/or immunological T cell memory play an important role.See also Samter, et al. (eds) Immunological Diseases vols. 1 and 2,Little, Brown and Co. Suppression of T cell activation, expansion,and/or cytokine release by the naturally occurring secreted form ofSLAM, which can be produced in large quantities by recombinant methods,or by blocking antibodies, should be effective in many disorders inwhich abnormal T cell responses are of importance.

The SLAM appears to be coexpressed with CD45RO, which is a marker forprimed, or memory, T cells. SLAM is also absent in the CD45RA cells,which represent the naive T cell subset. As such, the SLAM can alsoserve as a diagnostic marker for memory T cells.

Various abnormal conditions are known in each of the cell types shown topossess SLAM MRNA by Northern blot analysis. See Berkow (ed.) The MerckManual of Diagnosis and Therapy, Merck & Co., Rahway, N.J.; Thorn, etal. Harrison's Principles of Internal Medicine, McGraw-Hill, N.Y.; andWeatherall, et al. (eds.) Oxford Textbook of Medicine, Oxford UniversityPress, Oxford. Many other medical conditions and diseases involve Tcells or are T cell mediated, and many of these will be responsive totreatment by an agonist or antagonist provided herein. See, e.g., Stitesand Terr (eds; 1991) Basic and Clinical Immunology Appleton and Lange,Norwalk, Conn.; and Samter, et al. (eds) Immunological Diseases Little,Brown and Co. These problems should be susceptible to prevention ortreatment using compositions provided herein.

SLAM antibodies can be purified and then administered to a patient,veterinary or human. These reagents can be combined for therapeutic usewith additional active or inert ingredients, e.g., in conventionalpharmaceutically acceptable carriers or diluents, e.g., immunogenicadjuvants, along with physiologically innocuous stabilizers, excipients,or preservatives. These combinations can be sterile filtered and placedinto dosage forms as by lyophilization in dosage vials or storage instabilized aqueous preparations. This invention also contemplates use ofantibodies or binding fragments thereof, including forms which are notcomplement binding.

Drug screening using SLAM or fragments thereof can be performed toidentify compounds having binding affinity to or other relevantbiological effects on SLAM functions, including isolation of associatedcomponents. Subsequent biological assays can then be utilized todetermine if the compound has intrinsic stimulating activity and istherefore a blocker or antagonist in that it blocks the activity of theantigen. Likewise, a compound having intrinsic stimulating activity canactivate the signal pathway and is thus an agonist in that it simulatesthe activity of SLAM. This invention further contemplates thetherapeutic use of blocking antibodies to SLAM as antagonists and ofstimulatory antibodies, e.g., A12, as agonists. This approach should beparticularly useful with other SLAM species variants.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.)(1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics,8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17thed. (1990), Mack Publishing Co., Easton, Pa. Methods for administrationare discussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 pM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or a slow release apparatus will often be utilized forcontinuous or long term administration. See, e.g., Langer (1990) Science249:1527-1533.

SLAM, fragments thereof, and antibodies to it or its fragments,antagonists, and agonists, may be administered directly to the host tobe treated or, depending on the size of the compounds, it may bedesirable to conjugate them to carrier proteins such as ovalbumin orserum albumin prior to their administration. Therapeutic formulationsmay be administered in any conventional dosage formulation. While it ispossible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationstypically comprise at least one active ingredient, as defined above,together with one or more acceptable carriers thereof. Each carriershould be both pharmaceutically and physiologically acceptable in thesense of being compatible with the other ingredients and not injuriousto the patient. Formulations include those suitable for oral, rectal,nasal, topical, or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. See, e.g., Gilman, et al.(eds.) (1990) Goodman and Gilman's: The Pharmacological Bases ofTherapeutics, 8th Ed., Pergamon Press; and Remington's PharmaceuticalSciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa.; Avis, etal. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications,Dekker, N.Y.; Lieberman, et al. (eds.) (1990) Pharmaceutical DosageForms: Tablets, Dekker, N.Y.; and Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disoerse Systems, Dekker, N.Y. The therapyof this invention may be combined with or used in association with otheragents.

Both the naturally occurring and the recombinant form of the SLAMs ofthis invention are particularly useful in kits and assay methods whichare capable of screening compounds for binding activity to the proteins.Several methods of automating assays have been developed in recent yearsso as to permit screening of tens of thousands of compounds in a shortperiod. See, e.g., Fodor, et al. (1991) Science 251:767-773, whichdescribes means for testing of binding affinity by a plurality ofdefined polymers synthesized on a solid substrate. The development ofsuitable assays can be greatly facilitated by the availability of largeamounts of purified, soluble SLAM as provided by this invention.

Other methods can be used to determine the critical residues in theSLAM-SLAM interactions. Mutational analysis can be performed, e.g., seeSomoza, et al. (1993) J. Exptl. Med. 178:549-558, to determine specificresidues critical in the interaction and/or signaling. Bothextracellular domains, involved in the homophilic interaction, or anintracellular domain, which provides interactions important inintracellular signaling, will be useful.

For example, antagonists can normally be found once the antigen has beenstructurally defined, e.g., by tertiary structure data. Testing ofpotential interacting analogs is now possible upon the development ofhighly automated assay methods using a purified SLAM. In particular, newagonists and antagonists will be discovered by using screeningtechniques described herein of particular importance are compounds foundto have a combined binding affinity for a spectrum of SLAM molecules,e.g., compounds which can serve as antagonists for species variants ofSLAM.

One method of drug screening utilizes eukaryotic or prokaryotic hostcells which are stably transformed with recombinant DNA moleculesexpressing a SLAM. Cells may be isolated which express a SLAM inisolation from other molecules. Such cells, either in viable or fixedform, can be used for standard binding partner binding assays. See also,Parce, et al. (1989) Science 246:243-247; and Owicki, et al. (1990)Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitivemethods to detect cellular responses.

Another technique for drug screening involves an approach which provideshigh throughput screening for compounds having suitable binding affinityto a SLAM and is described in detail in Geysen, European PatentApplication 84/03564, published on Sep. 13, 1984. First, large numbersof different small peptide test compounds are synthesized on a solidsubstrate, e.g., plastic pins or some other appropriate surface, seeFodor, et al. (1991). Then all the pins are reacted with solubilized,unpurified or solubilized, purified SLAM, and washed. The next stepinvolves detecting bound SLAM.

Rational drug design may also be based upon structural studies of themolecular shapes of the SLAM and other effectors or analogs. Effectorsmay be other proteins which mediate other functions in response tobinding, or other proteins which normally interact with SLAM. One meansfor determining which sites interact with specific other proteins is aphysical structure determination, e.g., x-ray crystallography or 2dimensional NMR techniques. These will provide guidance as to whichamino acid residues form molecular contact regions. For a detaileddescription of protein structural determination, see, e.g., Blundell andJohnson (1976) Protein Crystallography, Academic Press, New York.

IX. Kits

This invention also contemplates use of SLAM proteins, fragmentsthereof, peptides, and their fusion products in a variety of diagnostickits and methods for detecting the presence of another SLAM or bindingpartner. Typically the kit will have a compartment containing either adefined SLAM peptide or gene segment or a reagent which recognizes oneor the other, e.g., SLAM fragments or antibodies.

A kit for determining the binding affinity of a test compound to a SLAMwould typically comprise a test compound; a labeled compound, forexample a binding partner or antibody having known binding affinity forSLAM; a source of SLAM (naturally occurring or recombinant); and a meansfor separating bound from free labeled compound, such as a solid phasefor immobilizing the molecule. Once compounds are screened, those havingsuitable binding affinity to the antigen can be evaluated in suitablebiological assays, as are well known in the art, to determine whetherthey act as agonists or antagonists to the SLAM signaling pathway. Theavailability of recombinant SLAM polypeptides also provide well definedstandards for calibrating such assays.

A preferred kit for determining the concentration of, e.g., a SLAM in asample would typically comprise a labeled compound, e.g., bindingpartner or antibody, having known binding affinity for the antigen, asource of antigen (naturally occurring or recombinant) and a means forseparating the bound from free labeled compound, e.g., a solid phase forimmobilizing the SLAM. Compartments containing reagents, andinstructions, will normally be provided.

Antibodies, including antigen binding fragments, specific for the SLAMor fragments are useful in diagnostic applications to detect thepresence of elevated levels of SLAM and/or its fragments. Suchdiagnostic assays can employ lysates, live cells, fixed cells,immunofluorescence, cell cultures, body fluids, and further can involvethe detection of antigens related to the antigen in serum, or the like.Diagnostic assays may be homogeneous (without a separation step betweenfree reagent and antigen-binding partner complex) or heterogeneous (witha separation step). Various commercial assays exist, such asradioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA),enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique(EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and the like.See, e.g., Van Vunakis, et al. (1980) Meth Enzymol. 70:1-525; Harlow andLane (1980) Antibodies: A Laboratory Manual, CSH Press, NY; and Coligan,et al. (eds.) (1993) Current Protocols in Immunology, Greene and Wiley,NY.

Anti-idiotypic antibodies may have similar use to diagnose presence ofantibodies against a SLAM, as such may be diagnostic of various abnormalstates. For example, overproduction of SLAM may result in production ofvarious immunological reactions which may be diagnostic of abnormalphysiological states, particularly in proliferative cell conditions suchas cancer or abnormal activation or differentiation.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody or binding partner, or labeled SLAMis provided. This is usually in conjunction with other additives, suchas buffers, stabilizers, materials necessary for signal production suchas substrates for enzymes, and the like. Preferably, the kit will alsocontain instructions for proper use and disposal of the contents afteruse. Typically the kit has compartments for each useful reagent.Desirably, the reagents are provided as a dry lyophilized powder, wherethe reagents may be reconstituted in an aqueous medium providingappropriate concentrations of reagents for performing the assay.

Any of the aforementioned constituents of the drug screening and thediagnostic assays may be used without modification or may be modified ina variety of ways. For example, labeling may be achieved by covalentlyor non-covalently joining a moiety which directly or indirectly providesa detectable signal. In any of these assays, the binding partner, testcompound, SLAM, or antibodies thereto can be labeled either directly orindirectly. Possibilities for direct labeling include label groups:radiolabels such as ¹²⁵ I, enzymes (U.S. Pat. No. 3,645,090) such asperoxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat.No. 3,940,475) capable of monitoring the change in fluorescenceintensity, wavelength shift, or fluorescence polarization. Possibilitiesfor indirect labeling include biotinylation of one constituent followedby binding to avidin coupled to one of the above label groups.

There are also numerous methods of separating the bound from the freeSLAM, or alternatively the bound from the free test compound. The SLAMcan be immobilized on various matrixes followed by washing. Suitablematrixes include plastic such as an ELISA plate, filters, and beads.See, e.g., Coligan, et al. (eds.) (1993) Current Protocols inImmunology, Vol. 1, Chapter 2, Greene and Wiley, NY. Other suitableseparation techniques include, without limitation, the fluoresceinantibody magnetizable particle method described in Rattle, et al. (1984)Clin. Chem. 30:1457-1461, and the double antibody magnetic particleseparation as described in U.S. Pat. No. 4,659,678.

Methods for linking proteins or their fragments to the various labelshave been extensively reported in the literature and do not requiredetailed discussion here. Many of the techniques involve the use ofactivated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence of aSLAM. These sequences can be used as probes for detecting levels of theSLAM message in samples from patients suspected of having an abnormalcondition, e.g., cancer or developmental problem. The preparation ofboth RNA and DNA nucleotide sequences, the labeling of the sequences,and the preferred size of the sequences has received ample descriptionand discussion in the literature. See, e.g., Langer-Safer, et al. (1982)Proc. Nat'l. Acad. Sci. 79:4381-4385; Caskey (1987) Science 236:962-967;and Wilchek et al. (1988) Anal. Biochem. 171:1-32.

Diagnostic kits which also test for the qualitative or quantitativepresence of other markers are also contemplated. Diagnosis or prognosismay depend on the combination of multiple indications used as markers.Thus, kits may test for combinations of markers. See, e.g., Viallet, etal. (1989) Progress in Growth Factor Res. 1:89-97.

The binding of SLAM-Ig to SLAM transfected L cells demonstrates thatSLAM can interact with itself as a ligand. Native gel electrophoresis ofpurified SLAM-Ig indicated directly, with the existence of highmolecular weight forms, that SLAM-Ig molecules were also capable ofhomophilic interaction in solution. Although monomeric and dimeric formsof SLAM-Ig were predominant on the native gel they were not distinctbands, indicative of a fairly weak molecular interaction susceptible todissociation during electrophoresis. Indeed the level of SLAM-Ig bindingto SLAM expressing L cells was lower than that observed using anequivalent concentration of monoclonal antibody, suggesting thatSLAM-SLAM interaction is weaker than the interaction of the mAb A12 withSLAM. Interactions between other Ig superfamily members aresubstantially weaker than the interaction of antibodies (van der Merweand Barclay (1994) TIBS 19:354-358). Consistent with SLAM being a ligandfor itself, SLAM-Ig binding was observed on T-cell clones andEBV-transformed B cells, both cell types which express significantlevels of SLAM. The levels of SLAM on CD45RO⁺ T cells from PBMCcorrelated with SLAM-Ig binding levels following activation. These datado not exclude that there may be another ligand for SLAM, but there isno evidence for another ligand since no SLAM-negative cell-type testedso far has shown SLAM-Ig binding, and when SLAM-Ig binding was observedit was proportional to the level of SLAM expression.

Consistent with the biochemical evidence that SLAM is a natural ligandfor itself, L cells transfected with SLAM could provide a directco-stimulatory signal for CD4⁺ T-cell clones. Engagement of SLAM withthe mAb A12 provides a significant co-stimulatory signal for T-cellactivation. As observed with the agonistic mAb A12, activation of CD4⁺T-cell clones via SLAM expressed on L cells, in combination withanti-CD3, leads to large increases in proliferation. Co-stimulation ofproliferation with suboptimal doses of anti-CD3 was observed withSLAM-transfectants. The stimulation provided by SLAM transfected L cellswas substantial enough to lead directly to T-cell proliferation in theabsence of other stimuli. In this respect, the direct stimulatory signalprovided by SLAM expressed on L cells is unique, and is not observedeven for the classical co-stimulatory molecules B7 (Jenkins and Johnson(1993) Curr. Opin. Immunol. 5:361-367) and B70 (Azuma, et al. (1993)Nature 366:76-79).

The ligand for B7 is CD28, and anti-CD28 mAbs do not directly stimulateproliferation of T-cell clones. However, the anti-SLAM mAb A12, or itsF(ab)₂ fragments can directly induce T-cell proliferation. Theconsequences of engagement of SLAM on T-cell clones by SLAM ontransfected L cells, or by mAb A12 or its F(ab)₂ fragments areconcordant. Thus, direct engagement of SLAM, without the involvement ofother molecules in the interaction, is sufficient to induce thefunctional effects observed. This does not preclude the likelyinteraction of SLAM with signal-transducing molecules, or diminish theimportance of other cell-surface molecule interactions in achieving themost potent functional effects of SLAM engagement, such as thetremendous co-stimulatory effects via SLAM on T cells stimulated in anantigen-specific manner.

The SLAM gene was localized to the interface of bands q21.3 and q22 onhuman chromosome 1. This region of chromosome 1 appears to be animportant locus for genes involved in cell-cell interactions. The genesfor selectins (Watson, et al. (1990) J. Exp. Med. 172:263-272),molecules involved in leukocyte adhesion and trafficking, also localizeto lq22-23. Another gene at this locus (lq21.3-23) is the gene formyelin Po (Pham-Dinh, et al. (1993) Hum. Mol. Genet. 2:2051-2054), themost abundant protein in myelin (Filbin, et al. (1990) Nature344:871-872). Like SLAM, myelin Po is a member of the Ig-superfamily(Williams and Barclay (1988) Annu. Rev. Immunol. 6:381-405) and alsointeracts homophilically. Normal myelin structure relies upon theself-interaction of myelin Po, and inherited mutations in myelin Po areresponsible for the Charcot-Marie-Tooth neuropathy, type lb (Kulkens, etal. (1993) Nat. Genet. 5:35-39; Hayasaka, et al. (1993) Nat. Genet.5:31-34). Many members of the Ig-superfamily interact heterophilicallywith related family members, prominent examples being CD2 with LFA-3(Selvaraj, et al. (1987) Nature 326:400-403) or CD48 (van der Merwe, etal. (1993) EMBO J. 12:4945-4954); CD28 with B7-1 (Linsley, et al. (1990)Proc. Natl. Acad. Sci. USA 87:5031-5035) or B7-2 (Freeman, et al. (1993)Science 262:909-911; Azuma, et al. (1993) Nature 366:76-79); and the TCRwith MHC class II (Matsui, et al. (1991) Science 254:1788-1791). Thatmany Ig-superfamily members can interact in this way may be the resultof evolution after gene duplication of a homophilically interactingprecursor (Williams and Barclay (1988) Annu. Rev. Immunol. 6:381-405).SLAM and myelin Po may have retained a primordial function ofIg-superfamily members to interact homophilically.

The gene for CD48 localizes to the same part of chromosome 1 as SLAM atlq21-23 (Staunton, et al. (1989) J. Exp. Med. 169:1087-1099). CD48,reported to be a weak ligand for CD2 (van der Merwe, et al. (1993) EMBOJ. 12:4945-4954), and 2B4, a signaling molecule expressed on murine NKcells and cytotoxic T cells (Mathew, et al. (1993) J. Immunol.151:5328-5337) for which a ligand has not been reported, are the mostclosely related molecules to SLAM. Interestingly, SLAM, CD48, and 2B4all have one V and one C domain and can be distinguished from othermembers of the Ig-superfamily by the conservation of the sequenceCXLXLXC, the second cysteine being the tether for the C-domain and thefirst cysteine a conserved residue probably between the V- andC-domains. CD48 and 2B4 have not yet been directly assessed for theirability to interact homophilically, however it has been reported that arecombinant soluble form CD48 tends to aggregate in solution. Therelatedness and chromosomal co-localization of CD48 and SLAM isindicative of evolutionary divergence following gene duplication.

Other large Ig-superfamily members with multiple domains have beenreported to interact homophilically, and these includeplatelet-endothelial cell adhesion molecule (CD31) (Watt, et al. (1993)Blood 82:2649-2663), neuron-glia cell adhesion molecule (Grumet andEdelman (1988) J. Cell Biol. 106:487-503), neuron-glia-related celladhesion molecule (Mauro, et al. (1992) J. Cell Biol. 119:191-202),neural cell adhesion molecule or CD56 (Rao, et al. (1992) J. Cell Biol.118:937-949), and the carcinoembryonic antigen (Zhou, et al. (1993) J.Cell Biol. 122:951-960).

An alternatively spliced form of SLAM lacking a 90 bp exon,corresponding to and precisely encompassing the transmembrane region ofSLAM encodes a secreted form of SLAM. This naturally produced moleculeexpressed by activated T cells may suppress T-cell function and may bepart of a negative feedback loop to attenuate, or locally restrict SLAMmediated activation upon cell-cell interaction. SLAM mediated T-cellactivation is resistant to cyclosporin, consistent with the inability ofanti-IL-2 antibodies to inhibit SLAM induced T-cell clone proliferation.Given the potent co-stimulatory effects of SLAM engagement on T-cellproliferation and Th1 cytokine production, the potentialimmunosuppressive activity of soluble SLAM may make it an effectiveadjunct for inhibiting ongoing immune responses relatively resistant tocyclosporin such as that seen in allograft rejection (Pereira, et al.(1990) J. Immunol. 144:2109-2116; Zeevi, et al. (1988) Hum. Immunol.21:143-153).

SLAM engagement has unique consequences for T-cell activation in termsof its ability to modulate cytokine production profiles toward a Th0/Th1subtype and, under some circumstances, to directly induce T-cellproliferation. The newly described SLAM appears to be a member of theIg-superfamily in addition to the TCR, CD28, CTLA-4, CD4, and CD2, andits engagement regulates T-cell responses. The presence of SLAM onlymphocytes indicates that activated lymphocytes themselves can providea significant co-stimulus. This is not unexpected, as the mostpredominant cell type in lymphoid organs are lymphocytes, which arestatistically ever present collaborators, and the major source ofautocrine T-cell growth factors such as IL-2. SLAM may not only providestrong co-stimulatory signals, but could also be involved in maintainingthe relative segregation and lymphocyte accumulation within lymphoidorgans. Most work on T-cell co-stimulation has focused on differentantigen-presenting cells and the molecules they express, particularly B7and B70, the ligands for CD28 and CTLA-4 (Jenkins (1994) Immunity1:443-446). B cells are an antigen-presenting cell which when activatedexpress SLAM, which may support B-T cell collaboration leading to Igproduction. Consistent with the co-stimulatory functions describedherein for SLAM, recent studies on CD28 deficient mice have invoked arole for other co-stimulatory molecules in T cell activation (Green, etal. (1994) Immunity 1:501-508; Shahinian, et al. (1993) Science261:609-612) and have indicated that co-stimulation provided by other Tcells can contribute to T cell activation (Green, et al. (1994) Immunity1:501-508; Jenkins (1994) Immunity 1:443-446) In addition to SLAM,activated human T cells do express MHC class II and B7 and have beenshown to be able to present antigen (Azuma, et al. (1993) J. Exp. Med.177:845-850), emphasizing the potential of interactions between T cells,which may alleviate the requirement for the constant presence ofantigen-presenting cells during the clonal expansion of T cells.Naturally produced soluble SLAM should provide a useful antagonist tofurther assess the importance of SLAM-SLAM interactions in thedevelopment of human immune reactions.

Anti-SLAM monoclonal antibodies inhibit IL-4 induced IgE synthesis,which indicates that signaling through SLAM either at the T helper cellor at the B cell levels, inhibits productive T-B cell interaction, whichresult in IL-4 driven IgE switching and IgE production. This effect canbe direct, e.g., through interactions between SLAM on T cells and SLAMon B cells, or indirect, e.g., by inducing cytokine production by theT-helper cell, which inhibits IL-4 driven IgE synthesis. Interferon-γ isthe primary example of such a cytokine.

These results also suggest that soluble forms of SLAM with agonistactivities may be able to prevent IL-4 and/or IL-13 driven IgE synthesisin atopic patients, and thereby will have therapeutic utility indownregulating IgE-mediated allergic diseases. In addition, the factthat engagement of SLAM induces preferentially Th1 cytokine production,SLAM agonists may have general clinical utility in redirecting Th2responses to Th1 responses in diseases in which a clear Th2 profile hasbeen implicated, such as allergy, certain autoimmune diseases, orcertain inflammatory diseases. This includes Hashimoto's thyroiditis.

On the other hand, SLAM antagonists will have an opposite effect; thatis, blocking of Th1 responses in the disease situations caused by Th1cells and Th1 cell derived cytokines, such as infectious diseases,including, e.g., tuberculosis and leprosy, or autoimmune diseases, e.g.,rheumatoid arthritis and autoimmune uveitis.

These therapeutic reagents will be useful also in modulating suchresponses as to parasitic infections, to modulate a vaccine reaction, orin

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

EXAMPLES General Methods

Some of the standard methods are described or referenced, e.g., inManiatis, et al. (1982) Molecular Clonina, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed.), vols 1-3, CSHPress, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology, Greene and Wiley, New York; Innis, etal. (eds.)(1990) PCR Protocols: A Guide to Methods and Applications,Academic Press, N.Y. Methods for protein purification include suchmethods as ammonium sulfate precipitation, column chromatography,electrophoresis, centrifugation, crystallization, and others. See, e.g.,Ausubel, et al. (1987 and periodic supplements); Deutscher (1990) "Guideto Protein Purification" in Methods in Enzymology vol. 182, and othervolumes in this series; and manufacturer's literature on use of proteinpurification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad,Richmond, Calif. Combination with recombinant techniques allow fusion toappropriate segments, e.g., to a FLAG sequence or an equivalent whichcan be fused via a protease-removable sequence. See, e.g., Hochuli(1989) Chemische Industrie 12:69-70; Hochuli (1990) "Purification ofRecombinant Proteins with Metal Chelate Absorbent" in Setlow (ed.)Genetic Engineering, Principle and Methods 12:87-98, Plenum Press, N.Y.;and Crowe, et al. (1992) OIAexDress: The Hiah Level Expression & ProteinPurification System QUIAGEN, Inc., Chatsworth, Calif. Cell culturetechniques are described in Doyle, et al. (eds.) (1994) Cell and TissueCulture: Laboratory Procedures, John Wiley and Sons, NY.

FACS analyses are described in Melamed, et al. (1990) Flow Cytometry andSorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988) Practical FlowCytometry Liss, New York, N.Y.; and Robinson, et al. (1993) Handbook ofFlow Cytometry Methods Wiley-Liss, New York, N.Y. Fluorescent labelingof appropriate reagents was performed by standard methods.

Example 1:

Preparation of mAb

The anti-SLAM monoclonal antibody A12 (IgGl) was generated in a fusionof splenocytes from a BALB/c mouse immunized with peripheral bloodmononuclear cells activated for 5 hours with 12-0-tetradecanoyl-13Acetate (TPA) (1 ng/ml) and the Ca²⁺ ionophore A23187 (500 ng/ml)(Calbiochem-Behring Corporation).

Standard procedures were used to screen for appropriate producingclones, and the A12 hybridoma was clonally isolated and subjected tonormal analysis, e.g., determination of producing capacity andimmunoglobulin type. The A12 hybridoma cell line was deposited with theATCC on Nov. 10, 1994, and has been assigned ATCC Accession NumberHB11760.

Example 2:

Cloning of Human SLAM

COS-7 cells were transfected by electroporation as described in Cocks,et al. (1993) Int. Immunol. 5:657-663, with an A10 CD8⁺ T-cell libraryDNA prepared as described in McKinney and Parkinson (1987) J. Immunol.Methods 96:271-278. Transfected cells were stained with FITC-conjugatedanti-SLAM mAb A12 and sorted with a FACStar plus (Becton Dickinson) cellsorting instrument. Plasmid DNA was isolated from sorted cells using aWizard miniprep kit (Promega Corporation). Plasmid DNA was transformedin Escherichia coli (ElectroMAX, BRL) by electroporation foramplification and then introduced into COS-7 cells. After two rounds ofsorting SLAM cDNA clones were enriched to 45% of the total cDNA clones.A 1.8 kb insert in one of these clones (pSURslaml) was sequenced in bothstrands using the dideoxy chain termination method. This plasmid wasdeposited with the ATCC on Nov. 10, 1994, and has been assigned ATCCAccession number 69713. Other cDNA clones encoding SLAM variants wereisolated and characterized using standard methods. In particular,constructs encoding an extracellular portion of SLAM, or anintracellular portion were prepared by use of appropriate PCR primersand pSURslam1 as template.

Example 3:

Cloning of Mouse SLAM

The mouse SLAM CDNA was cloned from an early thymocyte CDNA library,i.e., αβ, CD4⁻, CD8⁻ thymocytes, using DNA representing theextracellular domain of human SLAM as a hybridization probe.

Thymocytes were isolated and stained with primary antibody for 30 min at4° C., washed twice, and then incubated with FITC-conjugated secondaryantibody for 30 min at 4° C. before washing three times. Freshlyisolated thymocytes were stained with anti-SLAM monoclonal antibody orIgG, followed by an FITC-conjugated sheep anti-mouse antibody. Cellswere assessed for staining using a FACScan (Becton-Dickinson)instrument.

Example 4:

Preparation of Antibodies to Human SLAM

C3H mice were immunized with L-cells stably transfected with pSURslam1.Hybridomas were generated by fusing splenocytes with the NJ1 myelomaline. Detection of the hybridoma cells producing appropriate monoclonalantibodies to human SLAM was by indirect immunofluorescence and flowcytometry. The hybridoma supernatants were screened for reactivity withpSURslam1 transfected L cells, compared to untransfected L cells ascontrol.

Example 5:

Preparation of Antibodies to Mouse SLAM

Rats were immunized with 10⁷ COS cells transfected with pMSLAM1.Hybridomas were prepared by fusing rat popliteal lymph node cells withmouse myeloma cells. Polyclonal serum was also isolated from the rats.

Example 6:

Immunoprecipitation of Human SLAM

Cell-surface proteins of the Th0 T-cell clone B21 were radiolabeled with¹²⁵ I-Na (Amersham) using the lactoperoxidose-catalyzed reaction. SLAMwas immuno-precipitated using Pansorbin™ (Calbiochem) coated with rabbitanti-mouse Ig and the anti-SLAM anti-serum. The immunoprecipitates wererun on a 10% acrylamide minigel (ISS) under reducing conditions, and thedried gel was scanned and analyzed with a Phosphorimager (MolecularDynamics).

The natural SLAM migrated in a diffuse manner characteristic ofglycoproteins, at a mobility characteristic of about 70 kd. If the SLAMwas treated overnight with 1.2 μl N-glycanase (Genzyme), the proteinmigrated at a mobility characteristic of a protein of about 40 kd.

Example 7:

SLAM Expression on Human PBMC

SLAM expression on human PBMC is induced by exposure of the cells toanti-CD3 antibodies for differing time periods. Peripheral bloodlymphocytes were incubated with anti-CD3 antibodies (1 μg/ml) for 0, 1,2, 4, 8, or 24 hours. RNA was extracted and subjected to Northernanalysis using SLAM and actin probes, successively. For PCR analysis,appropriate primers were selected for SLAM and for HPRT. 5 ng of CDNAprimers was subject to 30 cycles of PCR. The actin signal serves as anormalization factor.

A 4 kb species is apparent at the 2 and 4 hour time points, and is muchless detectable at the 0, 1, 8, and 24 hour time points. A 2 kb speciesis less detectable at the 0 and 1 hour time points, is high at the 2hour point, decreases at the 4 hour, and stabilizes at the 8 and 24 hourpoints.

Example 8:

Surface expression of SLAM on mononuclear cells and fetal thymocytes

For FACS analysis, peripheral blood mononuclear cells from a healthydonor were incubated for 6 h with or without TPA and A23187 Ca² ⁺ionophore and stained with anti-CD3 cychrome conjugated (Pharmingen),PE-conjugated A12 mAb, and FITC-labeled CD45RO (Pharmingen). Inaddition, fetal thymocytes were stained for 30 minutes withPE-conjugated A12 and FITC-conjugated anti-CD3 (Becton Dickinson) andanalyzed with a FACScan (Becton Dickinson).

Unstimulated peripheral blood T cells and activated T cells (CD3⁺ cells)were stained with mAbs to CD45RO and A12. Similarly, fetal thymocyteswere stained with anti-CD3 and A12.

The unstimulated T cells had two significant subpopulations: one withlittle or no SLAM and no CD45RO, this comprising about 49% of the cells;and one with low SLAM and high CD45RO, this subpopulation comprisingabout 51% of the cells. The CD45RO is a marker for memory T cells, andthe SLAM seems to positively correlate with its expression. Naive Tcells, which are CD45RO⁻, also lack SLAM. The SLAM seems to be a usefulmarker for a memory T cell phenotype.

The activated T cells had two major subpopulations: both with high SLAM,but one had low CD45RO, this making up about 46% of the cells, and thesecond had high CD45RO. A minor subpopulation, about 4% of the cells,expressed neither CD45RO nor SLAM.

Fetal thymocytes had a pattern which seems to suggest a developmentalprogression. There is a minor subpopulation of cells, about 2%, whichexhibit neither SLAM nor CD3. About 13% of the cells, presumably earlydevelopment cells, which exhibit low CD3, and high SLAM. About 80% ofthe cells, presumably at an intermediate stage of development, whichexpress both CD3 and SLAM. A small subpopulation, about 5% of the cells,are mature thymocytes which exhibit low SLAM but high CD3. This probablyreflects a progression of SLAM expression with thymocyte maturation. Atthe earliest maturation stages, SLAM is highly expressed, but eventuallydisappears.

Example 9:

Cellular Expression of Human SLAM

RNA from various cells and tissues was subject to reverse transcriptionand PCR using SLAM specific primers. See Table 4 for tissue distributionof human SLAM.

Example 10:

Cellular Expression of Mouse SLAM1

A probe specific for DNA encoding a portion of the extracellular domainof mouse SLAM1 was used to determine tissue distribution of the antigen.A 600 bp probe DNA for murine SLAM was generated by a XhoI/PstI limitdigest of the plasmid pMSLAM1 (containing the mouse SLAM CDNA) andpurified after gel electrophoresis using a Promega (Madison, Wis.) DNAClean Up system. All probes were labeled by random priming. The multipletissue Northern blot was purchased from Clontech and probed using QuickHyb (Stratagene, La Jolla, Calif.).

The results showed that SLAM was expressed far more abundantly in spleenthan in heart, brain, lung, liver, skeletal muscle, kidney, or testes.Testes appeared to have more expression than other tissues but not asmuch as thymus. Although thymus was not one of the tissues on theNorthern blot, SLAM must be expressed there. The mouse SLAM cDNA wascloned from αβ, CD4⁻, CD8⁻ thymocytes and, in addition, a monoclonalantibody recognizing mouse SLAM bound specifically to 90% of freshlyisolated thymocytes. The frequency of SLAM clones in the thymocytelibrary was about 1 in 5000.

Example 11:

Species Distribution of SLAM Homologues

DNA was obtained from the various species, digested with EcoRI,electrophoresed, blotted, and transferred, then hybridized with a ³² plabeled human SLAM probe at 68° C. inclusive of nucleotides 291-901. Theblot was washed in 0.2×SSC at 60° C. Southern analysis of genomic DNAfrom different species indicated that the SLAM gene is well conservedamong mammals, e.g., human, monkey, mouse, dog, cow, rabbit, rabbit, butwas not detected in chicken or yeast. It was also not detected in rat,but no positive control was provided.

Example 12:

Enhancement of antigen-induced cytokine production by T-cell clonesso-stimulated with the anti-SLAM antibody A12

The indicated T cell clones, including the CD4⁺ T cell clones MoT72(Th2) and MoT81 (Th0) specific for tetanus toxoid, were cultured insimilar conditions as for the proliferative assays, with the followingmodifications: cultures were performed in 24 well plates culturing 10⁶ Tcells with 10⁶ irradiated autologous EBV-transformed B cells, antigen,and mAbs as described for the proliferative assays, in 1 ml Yssel'smedium. The supernatants were harvested 24 hours later and the cytokinecontent was determined by ELISA as described by Chretien, et al. (1989)J. Immunol. Methods 117:67-81; or Favre, et al. (1989) Mol. Immunol.26:17-25. The CD4⁺ T cell clones MOT72 (Th2) and MoT81 (Th0) arespecific for tetanus toxoid, and were cultured as described. See Table5. The mAbs used in this and the costimulation functional studies werepurified from ascites by caprilic acid fractionation, see McKinney andParkinson (1987) J. Immunol. Methods 96:271-278, followed by ammoniumsulphate precipitation. F(Ab')₂ were produced by standard methodsdigesting the mAbs with pepsin. The control mAbs used were IgGl fromMOPC-21 and IgGl control mAb (Pharmingen).

                  TABLE 5                                                         ______________________________________                                        Cytokine production, IFN-γ or pg/ml                                         Th type/cell line                                                                         no antibody  control Ab                                                                           A12 Ab                                    ______________________________________                                        IFN-γ                                                                       Th2/NP12     962          902    8303                                       Th2/NP44 1073 1319  7660                                                      Th2/MoT72  496  170  8585                                                     Th0/ChT38 5207 7463 20569                                                     Tho/MoT81 5423 6596 18176                                                     Th1/HY06 5982 5904 21946                                                      Th1/TA20 8374 8070 15414                                                    IL-4                                                                              Th2/NP12    6636         6486   11104                                       Th2/NP44 11617   11738  10373                                                 Th2/MoT72 8805 8542 16548                                                     Th0/ChT38 12907  10102  15039                                                 Tho/MoT81 8455 8451 11070                                                     Th1/HY06  48  40   90                                                         Th1/TA20  62  69   97                                                       ______________________________________                                    

Example 13:

Costimulatory Activity for T Cell Activation

Peripheral blood mononuclear cells (10⁵ /well) from recently boosteddonors were stimulated with 1 μg/ml of tetanus toxoid or purifiedprotein derivative (PPD) in flat-bottom 96-well plates in 200 μl Yssel'smedium in triplicate wells. The cultures were harvested five days later.1 μCi of ³ H-TdR was added to each well in the last 16 h of culture, andproliferation was measured by ³ H-Tdr uptake using a β-counter.

The following CD4⁺ T cell clones were used: Th0: B21 (Bacchetta, et al.(1990) J. Immunol. 144:902-908); MoT72 specific for tetanus toxoidfragment 947-960, and ChT38 specific for tetanus toxoid fragment 16-35(prepared according to Carballido, et al. (1993) J. Immunol,150:3582-3591. Th1: HY-06 (Haanen, et al. (1991) J. Exp. Med.174:583-592) specific for heat shock protein; TA20 and TA23 specific forpurified protein derivative (PPD). Th2: NP12 and NP44 (Th2) specific forthe Der-pl (Yssel, et al. (1992) J. Immunol. 148:738-745). All T cellclones were harvested 5-7 days following restimulation with PHA andirradiated PBMC as feeder cells and cultured in Yssel's medium (5×10⁴/well) in the presence or absence of specific antigen (1 μg/ml) ortetanus peptides (100 ng/ml), and 2.5×10⁵ autologous irradiated (5000rads) EBV-transformed B cells and mAbs as indicated. Proliferation wasmeasured 3 days later.

Direct induction of T cell clone proliferation by the anti-SLAM mAb A12:The T cell clones B21 and ChT38 were cultured in Yssel's medium in thepresence or absence of mAbs and their F(Ab')₂. Proliferation wasmeasured 3 days later. Dose dependent proliferation of the two celllines was observed.

Antigen-specific T-cell proliferation of peripheral blood lymphocytes isenhanced by the anti-SLAM antibody A12: Fab fragments of A12 induced adose dependent proliferation. PBMC from immunized donors were stimulatedwith tetanus toxoid or purified protein derivative, with or without mAbfragments.

Co-stimulation of antigen-induced T-cell clone proliferation by A12antibody: T cell clones NP12, AR142, ChT38, or HY06, were stimulatedwith their specific antigen with or without mAbs. All results areconsistent with an interpretation that either A12, or Fab fragments, caninduce proliferation in a dose dependent manner.

Co-stimulation of anti-CD3-induced T-cell clone proliferation by A12antibody: The T cell clones B21 or TA20 were stimulated with anti-CD3mAb in the presence or absence of A12 mAb or control IgGl mAb. In eachcase, there appeared a dose dependent proliferation with the A12, butnot with control antibody. The proliferation was also dependent upon theanti-CD3 amount.

Example 14:

Preparation of SLAM-Ig fusion

In order to identify a potential ligand for the T-cell co-stimulatorymolecule SLAM , a recombinant protein (SLAM-Ig) comprising the entireextracellular domain of SLAM fused to the Fc portion of human IgG wasgenerated. SLAM-Ig was made by fusing DNA encoding SLAM to DNA encodingthe Fc portion of IgG. DNA encoding the extracellular domain of SLAM wasgenerated by PCR using the plasmid pSURslaml as template and appropriateprimers. After digestion with XhoI the fragment was fused to cDNAencoding the Fc proportion of the IgGl heavy chain. The SLAM-Igexpression vector was transfected into COS cells and SLAM-Ig affinitypurified from the supernatants using protein G-sepharose (Sigma).

Example 15:

SLAM-Ig binds to SLAM expressed on the cell surface

Recombinant SLAM-Ig was effective in neutralizing the SLAM-specificmonoclonal antibody A12, indicating that SLAM-Ig had retained a nativeconformation similar to transmembrane SLAM. Fluorescein conjugatedSLAM-Ig was used for fluorocytometric analysis of various cell types anddid not bind to many cell types tested. However, SLAM-Ig did bind tocell types which have been shown to express SLAM.

Example 16:

Intramolecular interaction of SLAM-Ig

The T-cell clones B21 (Bacchetta, et al. (1990) J. Immunol. 144:902-908)and HY06 (Haanen, et al. (1991) J. Exp. Med. 174:583-592) have beendescribed. Thymic epithelial cell lines were generated as described byGaly and Spits (1991) J. Immunol. 147:3823-3830, by culture from fetalthymus and the lines TEC, TEC, U937 have been described also by Galy andSpits (1991). L cells carried in RPMI were stably transfected withpSURslam1. Monocytes were purified by negative depletion, and CD32 Lcells were provided by Dr. K. Moore (DNAX, Palo Alto). PBMC were freshlyisolated from peripheral blood by centrifugation over ficoll(Histopaque-1077, Sigma).

SLAM-Ig did bind to L cells transfected with SLAM (SLAM/L cells), andnot to untransfected L cells, indicating that SLAM interactshomophilically. The binding of SLAM-Ig to SLAM transfectants is specificfor the SLAM portion of the molecule and not the Ig, as the staining wasperformed in the presence of excess IgG in the 30% human serum added.Furthermore, SLAM transfectants were not stained by other Fc containingmolecules such as CD40-Ig. The binding of SLAM-Ig was about 5-fold lowerthan the binding to SLAM/L cells observed using an equivalentconcentration of the mAb A12. The interaction of SLAM-Ig with cellsurface SLAM could be specifically inhibited by an excess of amonoclonal antibody to SLAM. SLAM-Ig binding to transfected cells wasnot inhibited by EDTA.

The A12 anti-SLAM mAb has been described. Phycoerythrin conjugatedCD45RO and CD3 mAbs were purchased from Becton-Dickinson. Cells stainedwith mAbs, SLAM-Ig or CD40-Ig were washed three times with PBS, 2% FCSand analyzed using a FACScan (Becton-Dickinson).

Fluorescein conjugated SLAM-Ig was used for fluorocytometric analysis ofvarious cell types and did not bind to many cell types tested, includingmonocytes or thymic epithelial cell lines. However, SLAM-Ig did bind toEBV-transformed B-cell clones and CD4⁺ T-cell clones, both cell typeswhich we have shown to express SLAM. In no cell types tested did SLAM-Igbind to cells not expressing SLAM. In addition, the levels of SLAM-Igbinding co-modulated with SLAM expression on CD45RO⁺ T cells followingactivation with anti-CD3. The level of SLAM-Ig staining relative to A12staining on different cell-types was consistent with that observed onL-cell transfectants being 5-fold lower and Ca⁺⁺ independent.

Example 17:

Intermolecular interaction of SLAM-Ig

Gel electrophoresis was performed using gels purchased from IntegratedSeparation Systems and a BioRad multigel apparatus. SDS-electrophoresiswas performed under conditions described using a 10% gel and native gelelectrophoresis according to manufacturers instructions using a 2-25%gradient gel. Gels were stained with Coomassie Blue. MW standards werepurchased from Sigma.

Since a soluble form of SLAM (SLAM-Ig) can interact with cell surfaceSLAM, it was tested whether SLAM-Ig would interact homophilically, e.g.,self recognizing, in solution. Purified SLAM-Ig migrates to a positionconsistent with its size under SDS-gel electrophoresis and forms onediscrete band under reducing or non-reducing conditions. However SLAM-Igruns anomalously large under native gel electrophoresis, indicative ofaggregation of SLAM-Ig molecules in solution. CD40-Ig and other proteinsband sharply and according to their size, whereas under the sameconditions SLAM-Ig forms a smear beginning at its predicted size of160,000 without aggregation to over 500,000. Within this range ofmolecular weights there are two more predominant bands; one at ˜160,000and the other at ˜300,000 corresponding to one and two molecules ofSLAM-Ig, respectively. Gel filtration of SLAM-Ig confirmed the existenceof SLAM-Ig aggregates. Under these conditions, although the monomericform was more predominant, a peak corresponding to dimeric SLAM was alsoprominent among the higher molecular weight material.

Example 18:

Homophilic interaction of SLAM leads to T-cell activation

It was also shown that SLAM expressed on activated T cells is asignificant co-stimulatory molecule. Engagement of SLAM by the mAb A12leads to increases in T-cell proliferation and cytokine production. Thenatural ligand for SLAM should also provide such a co-stimulatorysignal. These results suggest that the natural ligand for SLAM is SLAMitself. Thus, the ability of surface SLAM to provide stimulatory signalsto T cells was tested. At suboptimal doses of anti-CD3, L cellsexpressing SLAM provided a direct co-stimulatory signal for T cells toproliferate, whereas, untransfected L cells were ineffective. SLAM/Lcells were also capable of directly supporting T-cell proliferation inthe absence of anti-CD3 or other stimulatory signals. This ability todirectly stimulate T cells in the absence of other stimuli distinguishesSLAM from other co-stimulatory molecules including LFA-3 (Bierer andHahn (1993) Semin. Immunol. 5:249-261), B7 (Jenkins and Johnson (1993)Curr. Opin. Immunol. 5:361-367), and B70 (Azuma, et al.. (1993) Nature366:76-79), each of which requires additional signals to induce T cellproliferation.

Since SLAM-SLAM interactions between L cells and T cells have clearfunctional effects, it was not surprising that L cells transfected withSLAM could be distinguished from untransfected L cells by at least threecriteria. First, SLAM⁺ L cells are resistant to detachment with EDTArequiring over 30 min at 37° C., compared with normal L cells and otherL cell transfectants, which become detached within 5 min. Secondly, theSLAM transfectants are strictly contact inhibited whereas, untransfectedL cells, although contact inhibited, do continue to proliferate to someextent after confluency has been reached. Thirdly, SLAM transfectantshave a more elongated morphology, evident in confluent monolayercultures where the cells are intertwined, in contrast to normal L cells,which have a more cobblestone appearance. Detached SLAM/L cells did notappear to adhere more readily in suspension.

Example 19:

T cell proliferation induced by SLAM-SLAM interaction is resistant tocyclosporin

T cell activation mediated via the TCR is inhibited by cyclosporin. Totest whether SLAM-mediated T cell activation was susceptible tocyclosporin, the T cell clone B21 was activated directly with SLAM/Lcells in the presence of various concentrations of cyclosporin. SLAM/Lcells were capable of directly supporting T-cell proliferation even inthe presence of 1 μg cyclosporin. Interestingly, cyclosporin actuallyenhanced T cell proliferation induced by homophilic interaction of SLAMat concentrations greater than 100 ng/ml. At 2 μg/ml, cyclosporinenhanced T cell proliferation induced by SLAM/L cells by 2 fold.

Example 20:

Chromosomal localization

The probe (pSURslaml) was nick-translated with biotin-14 dATP andhybridized in situ at a final concentration of 5 ng/μl to metaphasesfrom two normal males. The fluorescence in situ hybridization (FISH)method was modified from that described by Callen, et al. (1990). Ann.Genet. 33:219-221, in that chromosomes were stained before analysis withboth prodidium iodide (as counter stain) and DAPI (for chromosomeidentification). Images of metaphase preparations were captured by a CCDcamera and computer enhanced.

Twenty metaphases from the first normal male were examined forfluorescent signal. Nineteen of these metaphases showed signal on one orboth chromatids of chromosome 1 in the region lq21.2-lq23; 34% of thissignal was at lq21.3 and 59% was at lq22. This indicated a probablelocation close to the interface of these two bands. There was a total of4 non-specific background dots observed in these 20 metaphases. Asimilar result was obtained for hybridization of the probe to 20metaphases from the second normal male.

The gene maps to the same region as one which correlates with systemiclupus erythematosis susceptibility. The two genes may be the same, e.g.,SLAM reagents may be useful either as a direct therapeutic for thecondition, or the gene may be a useful genetic marker for mapping suchgene.

Example 21:

Kd of SLAM-SLAM interaction

The equilibrium constants for SLAM/SLAM interactions were analyze bysurface plasmon resonance using a BIAcore™ (Pharmacia) instrument. Ananti-SLAM Ab 7D4 was used.

Ab 7D4/SLAM-Ig and SLAM-Ig/SLAM-Ig binding kinetics and affinity weremeasured. About 8000 resonance units (RUs) of SLAM-Ig were covalentlyattached to the dextran matrix in the sensor chip via its lysines,according to the manufacturer's protocol (BIAcore™ manual, PharmaciaBiosensor). Buffer (phosphate-buffer saline, PBS, pH 7.0) was passedthrough the flow cell until all of the dissociable protein was removedand the baseline remained stable. Solutions containing variousconcentrations of the antibody 7D4 in PBS (ranging from 10 nM to 500 nM)were then passed through the flow cell. An increase in mass of proteinbound was observed, followed by a decrease when the protein solution wasreplaced with buffer. A non-linear data analysis protocol, O'Shannessy,et al. (1993) Anal. Biochem. 212:457-468, was used to analyse the datato determine the association (k_(a)) and dissociation (k_(d)) rateconstants. See Table 6. The equilibrium dissociation constant K_(d) wasthen calculated from the ratio k_(d) /k_(a).

SLAM-Ig/SLAM-Ig binding kinetics were measured in a similar manner.After immobilization of SLAM-Ig to the chip, solutions of SLAM-Ig atconcentrations ranging from 100 nM to 1500 nM were passed through theflow cell at a flow rate of 5 μl/min. From the association anddissociation phases, the corresponding rate constants and thus theequilibrium binding constant were obtained.

Both the k_(on) and k_(off) rates are slower than other cell-celladhesion interactions. The K_(d) is some 10-100 times higher than othercell-cell adhesion interactions, e.g., CD2 interaction with CD48 (about60-80 μm).

                  TABLE 6                                                         ______________________________________                                        Association and dissociation rate constants.sup.1 and the apparent             equilibrium constant K.sub.d for SLAM/SLAM and SLAM/Ab 7D4 interactions        Immobilized         k.sub.on                                                                                        surface Ligand (                                                             ×10.sup.4 M.sup.-1 s.sup.-1)                                            k.sub.off (s.sup.-1) K.sub.d           ______________________________________                                        SLAM-Ig Ab 7D4    1.32       5.5 × 10.sup.-5                                                                 4.2  nM                                    SLAM-Ig SLAM-Ig 1.2 0.011 0.92 mM                                           ______________________________________                                         .sup.1 The standard errors in the parameters were estimated to be:            k.sub.on, 20%; k.sub.off, 10%; k.sub.d , 24%.                            

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 12                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1716 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 61..1065                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - AGGCATCTGT GAGCAGCTGC CAGGCTCCGG CCAGGATCCC TTCCTTCTCC TC -            #ATTGGCTG     60                                                                 - - ATG GAT CCC AAG GGG CTC CTC TCC TTG ACC TT - #C GTG CTG TTT CTC        TCC      108                                                                    Met Asp Pro Lys Gly Leu Leu Ser Leu Thr Ph - #e Val Leu Phe Leu Ser            1               5 - #                 10 - #                 15              - - CTG GCT TTT GGG GCA AGC TAC GGA ACA GGT GG - #G CGC ATG ATG AAC TGC          156                                                                       Leu Ala Phe Gly Ala Ser Tyr Gly Thr Gly Gl - #y Arg Met Met Asn Cys                        20     - #             25     - #             30                  - - CCA AAG ATT CTC CGG CAG TTG GGA AGC AAA GT - #G CTG CTG CCC CTG ACA          204                                                                       Pro Lys Ile Leu Arg Gln Leu Gly Ser Lys Va - #l Leu Leu Pro Leu Thr                    35         - #         40         - #         45                      - - TAT GAA AGG ATA AAT AAG AGC ATG AAC AAA AG - #C ATC CAC ATT GTC GTC          252                                                                       Tyr Glu Arg Ile Asn Lys Ser Met Asn Lys Se - #r Ile His Ile Val Val                50             - #     55             - #     60                          - - ACA ATG GCA AAA TCA CTG GAG AAC AGT GTC GA - #G AAC AAA ATA GTG TCT          300                                                                       Thr Met Ala Lys Ser Leu Glu Asn Ser Val Gl - #u Asn Lys Ile Val Ser            65                 - # 70                 - # 75                 - # 80       - - CTT GAT CCA TCC GAA GCA GGC CCT CCA CGT TA - #T CTA GGA GAT CGC TAC          348                                                                       Leu Asp Pro Ser Glu Ala Gly Pro Pro Arg Ty - #r Leu Gly Asp Arg Tyr                            85 - #                 90 - #                 95              - - AAG TTT TAT CTG GAG AAT CTC ACC CTG GGG AT - #A CGG GAA AGC AGG AAG          396                                                                       Lys Phe Tyr Leu Glu Asn Leu Thr Leu Gly Il - #e Arg Glu Ser Arg Lys                       100      - #           105      - #           110                  - - GAG GAT GAG GGA TGG TAC CTT ATG ACC CTG GA - #G AAA AAT GTT TCA GTT          444                                                                       Glu Asp Glu Gly Trp Tyr Leu Met Thr Leu Gl - #u Lys Asn Val Ser Val                   115          - #       120          - #       125                      - - CAG CGC TTT TGC CTG CAG TTG AGG CTT TAT GA - #G CAG GTC TCC ACT CCA          492                                                                       Gln Arg Phe Cys Leu Gln Leu Arg Leu Tyr Gl - #u Gln Val Ser Thr Pro               130              - #   135              - #   140                          - - GAA ATT AAA GTT TTA AAC AAG ACC CAG GAG AA - #C GGG ACC TGC ACC TTG          540                                                                       Glu Ile Lys Val Leu Asn Lys Thr Gln Glu As - #n Gly Thr Cys Thr Leu           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - ATA CTG GGC TGC ACA GTG GAG AAG GGG GAC CA - #T GTG GCT TAC AGC        TGG      588                                                                    Ile Leu Gly Cys Thr Val Glu Lys Gly Asp Hi - #s Val Ala Tyr Ser Trp                          165  - #               170  - #               175              - - AGT GAA AAG GCG GGC ACC CAC CCA CTG AAC CC - #A GCC AAC AGC TCC CAC          636                                                                       Ser Glu Lys Ala Gly Thr His Pro Leu Asn Pr - #o Ala Asn Ser Ser His                       180      - #           185      - #           190                  - - CTC CTG TCC CTC ACC CTC GGC CCC CAG CAT GC - #T GAC AAT ATC TAC ATC          684                                                                       Leu Leu Ser Leu Thr Leu Gly Pro Gln His Al - #a Asp Asn Ile Tyr Ile                   195          - #       200          - #       205                      - - TGC ACC GTG AGC AAC CCT ATC AGC AAC AAT TC - #C CAG ACC TTC AGC CCG          732                                                                       Cys Thr Val Ser Asn Pro Ile Ser Asn Asn Se - #r Gln Thr Phe Ser Pro               210              - #   215              - #   220                          - - TGG CCC GGA TGC AGG ACA GAC CCC TCA GAA AC - #A AAA CCA TGG GCA GTG          780                                                                       Trp Pro Gly Cys Arg Thr Asp Pro Ser Glu Th - #r Lys Pro Trp Ala Val           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - TAT GCT GGG CTG TTA GGG GGT GTC ATC ATG AT - #T CTC ATC ATG GTG        GTA      828                                                                    Tyr Ala Gly Leu Leu Gly Gly Val Ile Met Il - #e Leu Ile Met Val Val                          245  - #               250  - #               255              - - ATA CTA CAG TTG AGA AGA AGA GGT AAA ACG AA - #C CAT TAC CAG ACA ACA          876                                                                       Ile Leu Gln Leu Arg Arg Arg Gly Lys Thr As - #n His Tyr Gln Thr Thr                       260      - #           265      - #           270                  - - GTG GAA AAA AAA AGC CTT ACG ATC TAT GCC CA - #A GTC CAG AAA CCA GGT          924                                                                       Val Glu Lys Lys Ser Leu Thr Ile Tyr Ala Gl - #n Val Gln Lys Pro Gly                   275          - #       280          - #       285                      - - CCT CTT CAG AAG AAA CTT GAC TCC TTC CCA GC - #T CAG GAC CCT TGC ACC          972                                                                       Pro Leu Gln Lys Lys Leu Asp Ser Phe Pro Al - #a Gln Asp Pro Cys Thr               290              - #   295              - #   300                          - - ACC ATA TAT GTT GCT GCC ACA GAG CCT GTC CC - #A GAG TCT GTC CAG GAA         1020                                                                       Thr Ile Tyr Val Ala Ala Thr Glu Pro Val Pr - #o Glu Ser Val Gln Glu           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - ACA AAT TCC ATC ACA GTC TAT GCT AGT GTG AC - #A CTT CCA GAG AGC             1065                                                                      Thr Asn Ser Ile Thr Val Tyr Ala Ser Val Th - #r Leu Pro Glu Ser                               325  - #               330  - #               335              - - TGACACCAGA GACCAACAAA GGGACTTTCT GAAGGAAAAT GGAAAAACCA AA -             #ATGAACAC   1125                                                                 - - TGAACTTGGC CACAGGCCCA AGTTTCCTCT GGCAGACATG CTGCACGTCT GT -            #ACCCTTCT   1185                                                                 - - CAGATCAACT CCCTGGTGAT GTTTCTTCCA CATACATCTG TGAAATGAAC AA -            #GGAAGTGA   1245                                                                 - - GGCTTCCCAA GAATTTAGCT TGCTGTGCAG TGGCTGCAGG CGCAGAACAG AG -            #CGTTACTT   1305                                                                 - - GATAACAGCG TTCCATCTTT GTGTTGTAGC AGATGAAATG GACAGTAATG TG -            #AGTTCAGA   1365                                                                 - - CTTTGGGCAT CTTGCTCTTG GCTGGAACTG ATAATAAAAA TCAGACTGAA AG -            #CCAGGACA   1425                                                                 - - TCTGAGTACC TATCTCACAC ACTGACCACC AGTCACAAAG TCTGGAAAAG TT -            #TACATTTT   1485                                                                 - - GGCTATCTTT ACTTTGTTCT GGGAGCTGAT CATGATAACC TGCAGACCTG AT -            #CAAGCCTC   1545                                                                 - - TGTGCCTCAG TTTCTCTCTC AGGATAAAGA GTGAATAGAG GCCGAAGGGT GA -            #ATTTCTTA   1605                                                                 - - TTATACATAA AACACTCTGA TATTATTGTA TAAAGGAAGC TAAGAATATT AT -            #TTTATTTG   1665                                                                 - - CAAAACCCAG AAGCTAAAAA GTCAATAAAC AGAAAGAATG ATTTTGAGAA A - #               1716                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 335 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Asp Pro Lys Gly Leu Leu Ser Leu Thr Ph - #e Val Leu Phe Leu Ser        1               5 - #                 10 - #                 15              - - Leu Ala Phe Gly Ala Ser Tyr Gly Thr Gly Gl - #y Arg Met Met Asn Cys                   20     - #             25     - #             30                  - - Pro Lys Ile Leu Arg Gln Leu Gly Ser Lys Va - #l Leu Leu Pro Leu Thr               35         - #         40         - #         45                      - - Tyr Glu Arg Ile Asn Lys Ser Met Asn Lys Se - #r Ile His Ile Val Val           50             - #     55             - #     60                          - - Thr Met Ala Lys Ser Leu Glu Asn Ser Val Gl - #u Asn Lys Ile Val Ser       65                 - # 70                 - # 75                 - # 80       - - Leu Asp Pro Ser Glu Ala Gly Pro Pro Arg Ty - #r Leu Gly Asp Arg Tyr                       85 - #                 90 - #                 95              - - Lys Phe Tyr Leu Glu Asn Leu Thr Leu Gly Il - #e Arg Glu Ser Arg Lys                  100      - #           105      - #           110                  - - Glu Asp Glu Gly Trp Tyr Leu Met Thr Leu Gl - #u Lys Asn Val Ser Val              115          - #       120          - #       125                      - - Gln Arg Phe Cys Leu Gln Leu Arg Leu Tyr Gl - #u Gln Val Ser Thr Pro          130              - #   135              - #   140                          - - Glu Ile Lys Val Leu Asn Lys Thr Gln Glu As - #n Gly Thr Cys Thr Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ile Leu Gly Cys Thr Val Glu Lys Gly Asp Hi - #s Val Ala Tyr Ser        Trp                                                                                             165  - #               170  - #               175             - - Ser Glu Lys Ala Gly Thr His Pro Leu Asn Pr - #o Ala Asn Ser Ser His                  180      - #           185      - #           190                  - - Leu Leu Ser Leu Thr Leu Gly Pro Gln His Al - #a Asp Asn Ile Tyr Ile              195          - #       200          - #       205                      - - Cys Thr Val Ser Asn Pro Ile Ser Asn Asn Se - #r Gln Thr Phe Ser Pro          210              - #   215              - #   220                          - - Trp Pro Gly Cys Arg Thr Asp Pro Ser Glu Th - #r Lys Pro Trp Ala Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Tyr Ala Gly Leu Leu Gly Gly Val Ile Met Il - #e Leu Ile Met Val        Val                                                                                             245  - #               250  - #               255             - - Ile Leu Gln Leu Arg Arg Arg Gly Lys Thr As - #n His Tyr Gln Thr Thr                  260      - #           265      - #           270                  - - Val Glu Lys Lys Ser Leu Thr Ile Tyr Ala Gl - #n Val Gln Lys Pro Gly              275          - #       280          - #       285                      - - Pro Leu Gln Lys Lys Leu Asp Ser Phe Pro Al - #a Gln Asp Pro Cys Thr          290              - #   295              - #   300                          - - Thr Ile Tyr Val Ala Ala Thr Glu Pro Val Pr - #o Glu Ser Val Gln Glu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Thr Asn Ser Ile Thr Val Tyr Ala Ser Val Th - #r Leu Pro Glu Ser                         325  - #               330  - #               335              - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1852 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 61..954                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - TGGCATCTGT GAGCAGCTGC CAGGCTCCGG CCAGGATCCC TTCCTTCTCC TC -             #ATTGGCTG     60                                                                 - - ATG GAT CCC AAG GGG CTC CTC TCC TTG ACC TT - #C GTG CTG TTT CTC        TCC      108                                                                    Met Asp Pro Lys Gly Leu Leu Ser Leu Thr Ph - #e Val Leu Phe Leu Ser            1               5 - #                 10 - #                 15              - - CTG GCT TTT GGG GCA AGC TAC GGA ACA GGT GG - #G CGC ATG ATG AAC TGC          156                                                                       Leu Ala Phe Gly Ala Ser Tyr Gly Thr Gly Gl - #y Arg Met Met Asn Cys                        20     - #             25     - #             30                  - - CCA AAG ATT CTC CGG CAG TTG GGA AGC AAA GT - #G CTG CTG CCC CTG ACA          204                                                                       Pro Lys Ile Leu Arg Gln Leu Gly Ser Lys Va - #l Leu Leu Pro Leu Thr                    35         - #         40         - #         45                      - - TAT GAA AGG ATA AAT AAG AGC ATG AAC AAA AG - #C ATC CAC ATT GTC GTC          252                                                                       Tyr Glu Arg Ile Asn Lys Ser Met Asn Lys Se - #r Ile His Ile Val Val                50             - #     55             - #     60                          - - ACA ATG GCA AAA TCA CTG GAG AAC AGT GTC GA - #G AAC AAA ATA GTG TCT          300                                                                       Thr Met Ala Lys Ser Leu Glu Asn Ser Val Gl - #u Asn Lys Ile Val Ser            65                 - # 70                 - # 75                 - # 80       - - CTT GAT CCA TCC GAA GCA GGC CCT CCA CGT TA - #T CTA GGA GAT CGC TAC          348                                                                       Leu Asp Pro Ser Glu Ala Gly Pro Pro Arg Ty - #r Leu Gly Asp Arg Tyr                            85 - #                 90 - #                 95              - - AAG TTT TAT CTG GAG AAT CTC ACC CTG GGG AT - #A CGG GAA AGC AGG AAG          396                                                                       Lys Phe Tyr Leu Glu Asn Leu Thr Leu Gly Il - #e Arg Glu Ser Arg Lys                       100      - #           105      - #           110                  - - GAG GAT GAG GGA TGG TAC CTT ATG ACC CTG GA - #G AAA AAT GTT TCA GTT          444                                                                       Glu Asp Glu Gly Trp Tyr Leu Met Thr Leu Gl - #u Lys Asn Val Ser Val                   115          - #       120          - #       125                      - - CAG CGC TTT TGC CTG CAG TTG AGG CTT TAT GA - #G CAG GTC TCC ACT CCA          492                                                                       Gln Arg Phe Cys Leu Gln Leu Arg Leu Tyr Gl - #u Gln Val Ser Thr Pro               130              - #   135              - #   140                          - - GAA ATT AAA GTT TTA AAC AAG ACC CAG GAG AA - #C GGG ACC TGC ACC TTG          540                                                                       Glu Ile Lys Val Leu Asn Lys Thr Gln Glu As - #n Gly Thr Cys Thr Leu           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - ATA CTG GGC TGC ACA GTG GAG AAG GGG GAC CA - #T GTG GCT TAC AGC        TGG      588                                                                    Ile Leu Gly Cys Thr Val Glu Lys Gly Asp Hi - #s Val Ala Tyr Ser Trp                          165  - #               170  - #               175              - - AGT GAA AAG GCG GGC ACC CAC CCA CTG AAC CC - #A GCC AAC AGC TCC CAC          636                                                                       Ser Glu Lys Ala Gly Thr His Pro Leu Asn Pr - #o Ala Asn Ser Ser His                       180      - #           185      - #           190                  - - CTC CTG TCC CTC ACC CTC GGC CCC CAG CAT GC - #T GAC AAT ATC TAC ATC          684                                                                       Leu Leu Ser Leu Thr Leu Gly Pro Gln His Al - #a Asp Asn Ile Tyr Ile                   195          - #       200          - #       205                      - - TGC ACC GTG AGC AAC CCT ATC AGC AAC AAT TC - #C CAG ACC TTC AGC CCG          732                                                                       Cys Thr Val Ser Asn Pro Ile Ser Asn Asn Se - #r Gln Thr Phe Ser Pro               210              - #   215              - #   220                          - - TGG CCC GGA TGC AGG ACA GAC CCC TCA GAA AC - #A AAA CCA TGG GCA GTG          780                                                                       Trp Pro Gly Cys Arg Thr Asp Pro Ser Glu Th - #r Lys Pro Trp Ala Val           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - TAT GCT GGG CTG TTA GGG GGT GTC ATC ATG AT - #T CTC ATC ATG GTG        GTA      828                                                                    Tyr Ala Gly Leu Leu Gly Gly Val Ile Met Il - #e Leu Ile Met Val Val                          245  - #               250  - #               255              - - ATA CTA CAG TTG AGA AGA AGA GGT AAA ACG AA - #C CAT TAC CAG ACA ACA          876                                                                       Ile Leu Gln Leu Arg Arg Arg Gly Lys Thr As - #n His Tyr Gln Thr Thr                       260      - #           265      - #           270                  - - GTG GAA AAA AAA AGC CTT ACG ATC TAT GCC CA - #A GTC CAG AAA CCA GGT          924                                                                       Val Glu Lys Lys Ser Leu Thr Ile Tyr Ala Gl - #n Val Gln Lys Pro Gly                   275          - #       280          - #       285                      - - GAC ACT CAT CAT CAG ACT TCG GAC TTA TTC TA - #ATCCAGGA TGACCTTATT            974                                                                       Asp Thr His His Gln Thr Ser Asp Leu Phe                                           290              - #   295                                                 - - TTGAAATCCT TATCTTGACA TCTGTGAAGA CCTTTATTCA AATAAAGTCA CA -             #TTTTGACA   1034                                                                 - - TTCTGCGAGG GGCTGGAGCC GGGCCGGGGC GATGTGGAGC GCGGGCCGCG GC -            #GGGGCTGC   1094                                                                 - - CTGGCCGGTG CTGTTGGGGC TGCTGCTGGC GCTGTTAGTG CCGGGCGGTG GT -            #GCCGCCAA   1154                                                                 - - GACCGGTGCG GAGCTCGTGA CTGCGGGTCG GTGCTGAAGC TGCTCAATAC GC -            #ACCACCGG   1214                                                                 - - TGCGGCTGCA CTCGCACGAC ATCAAATACG GATCCGGCAG CGGCCAGCAA TC -            #GGTGACCG   1274                                                                 - - GCGTAGAGGT CGGAGCGACG AATAGCTACT GGCGGATCCG CGGCGGCTCG GA -            #GGGGGGTG   1334                                                                 - - CCCGCGCGGG TCCCCGGTGC GCTGCGGGCA GGCGGTGAGG TCACACATGT GC -            #TTACGGGC   1394                                                                 - - AAGAACCTGC ACACGCACCA CTTCCCGTCG CCGCTGTCCA ACAACCAGGA AG -            #TGAGTGCC   1454                                                                 - - AAAGGGGAAG ACGGCGAGGG CGACGACCTG GACCTATGGA CAGTGCGCTG CT -            #CTGCTCTG   1514                                                                 - - GACAGCACTG GGAGCGTGAG GCTGCTGTGG CGCCTTCCAG CATGTGGCAC CT -            #CTGTGGTT   1574                                                                 - - CCTGTCAGTC ACGGTAGCAG TATGGAAGCC CCATCCGTGG GCAGCATGAG GT -            #CCACGCAT   1634                                                                 - - GCCCAGTGCC AACACGCACA ATACGTGGAA GGCCATGGAA GGCATCTTCA TC -            #AAGCCTAG   1694                                                                 - - TGTGGAGCCC TCTGCAGGTC ACGATGAACT CTGAGTGTGT GGATGGATGG GT -            #GGATGGAG   1754                                                                 - - GGTGGCAGGT GGGGCGTCTG CAGGGCCACT CTTGGCAGAG ACTTTGGGTT TG -            #TAGGGGTC   1814                                                                 - - CTCAAGTGCC TTTGTGATTA AAGAATGTTG GTCTATGA      - #                      - #   1852                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 298 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Asp Pro Lys Gly Leu Leu Ser Leu Thr Ph - #e Val Leu Phe Leu Ser        1               5 - #                 10 - #                 15              - - Leu Ala Phe Gly Ala Ser Tyr Gly Thr Gly Gl - #y Arg Met Met Asn Cys                   20     - #             25     - #             30                  - - Pro Lys Ile Leu Arg Gln Leu Gly Ser Lys Va - #l Leu Leu Pro Leu Thr               35         - #         40         - #         45                      - - Tyr Glu Arg Ile Asn Lys Ser Met Asn Lys Se - #r Ile His Ile Val Val           50             - #     55             - #     60                          - - Thr Met Ala Lys Ser Leu Glu Asn Ser Val Gl - #u Asn Lys Ile Val Ser       65                 - # 70                 - # 75                 - # 80       - - Leu Asp Pro Ser Glu Ala Gly Pro Pro Arg Ty - #r Leu Gly Asp Arg Tyr                       85 - #                 90 - #                 95              - - Lys Phe Tyr Leu Glu Asn Leu Thr Leu Gly Il - #e Arg Glu Ser Arg Lys                  100      - #           105      - #           110                  - - Glu Asp Glu Gly Trp Tyr Leu Met Thr Leu Gl - #u Lys Asn Val Ser Val              115          - #       120          - #       125                      - - Gln Arg Phe Cys Leu Gln Leu Arg Leu Tyr Gl - #u Gln Val Ser Thr Pro          130              - #   135              - #   140                          - - Glu Ile Lys Val Leu Asn Lys Thr Gln Glu As - #n Gly Thr Cys Thr Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ile Leu Gly Cys Thr Val Glu Lys Gly Asp Hi - #s Val Ala Tyr Ser        Trp                                                                                             165  - #               170  - #               175             - - Ser Glu Lys Ala Gly Thr His Pro Leu Asn Pr - #o Ala Asn Ser Ser His                  180      - #           185      - #           190                  - - Leu Leu Ser Leu Thr Leu Gly Pro Gln His Al - #a Asp Asn Ile Tyr Ile              195          - #       200          - #       205                      - - Cys Thr Val Ser Asn Pro Ile Ser Asn Asn Se - #r Gln Thr Phe Ser Pro          210              - #   215              - #   220                          - - Trp Pro Gly Cys Arg Thr Asp Pro Ser Glu Th - #r Lys Pro Trp Ala Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Tyr Ala Gly Leu Leu Gly Gly Val Ile Met Il - #e Leu Ile Met Val        Val                                                                                             245  - #               250  - #               255             - - Ile Leu Gln Leu Arg Arg Arg Gly Lys Thr As - #n His Tyr Gln Thr Thr                  260      - #           265      - #           270                  - - Val Glu Lys Lys Ser Leu Thr Ile Tyr Ala Gl - #n Val Gln Lys Pro Gly              275          - #       280          - #       285                      - - Asp Thr His His Gln Thr Ser Asp Leu Phe                                      290              - #   295                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1020 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 61..975                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - AGGCATCTGT GAGCAGCTGC CAGGCTCCGG CCAGGATCCC TTCCTTCTCC TC -             #ATTGGCTG     60                                                                 - - ATG GAT CCC AAG GGG CTC CTC TCC TTG ACC TT - #C GTG CTG TTT CTC        TCC      108                                                                    Met Asp Pro Lys Gly Leu Leu Ser Leu Thr Ph - #e Val Leu Phe Leu Ser            1               5 - #                 10 - #                 15              - - CTG GCT TTT GGG GCA AGC TAC GGA ACA GGT GG - #G CGC ATG ATG AAC TGC          156                                                                       Leu Ala Phe Gly Ala Ser Tyr Gly Thr Gly Gl - #y Arg Met Met Asn Cys                        20     - #             25     - #             30                  - - CCA AAG ATT CTC CGG CAG TTG GGA AGC AAA GT - #G CTG CTG CCC CTG ACA          204                                                                       Pro Lys Ile Leu Arg Gln Leu Gly Ser Lys Va - #l Leu Leu Pro Leu Thr                    35         - #         40         - #         45                      - - TAT GAA AGG ATA AAT AAG AGC ATG AAC AAA AG - #C ATC CAC ATT GTC GTC          252                                                                       Tyr Glu Arg Ile Asn Lys Ser Met Asn Lys Se - #r Ile His Ile Val Val                50             - #     55             - #     60                          - - ACA ATG GCA AAA TCA CTG GAG AAC AGT GTC GA - #G AAC AAA ATA GTG TCT          300                                                                       Thr Met Ala Lys Ser Leu Glu Asn Ser Val Gl - #u Asn Lys Ile Val Ser            65                 - # 70                 - # 75                 - # 80       - - CTT GAT CCA TCC GAA GCA GGC CCT CCA CGT TA - #T CTA GGA GAT CGC TAC          348                                                                       Leu Asp Pro Ser Glu Ala Gly Pro Pro Arg Ty - #r Leu Gly Asp Arg Tyr                            85 - #                 90 - #                 95              - - AAG TTT TAT CTG GAG AAT CTC ACC CTG GGG AT - #A CGG GAA AGC AGG AAG          396                                                                       Lys Phe Tyr Leu Glu Asn Leu Thr Leu Gly Il - #e Arg Glu Ser Arg Lys                       100      - #           105      - #           110                  - - GAG GAT GAG GGA TGG TAC CTT ATG ACC CTG GA - #G AAA AAT GTT TCA GTT          444                                                                       Glu Asp Glu Gly Trp Tyr Leu Met Thr Leu Gl - #u Lys Asn Val Ser Val                   115          - #       120          - #       125                      - - CAG CGC TTT TGC CTG CAG TTG AGG CTT TAT GA - #G CAG GTC TCC ACT CCA          492                                                                       Gln Arg Phe Cys Leu Gln Leu Arg Leu Tyr Gl - #u Gln Val Ser Thr Pro               130              - #   135              - #   140                          - - GAA ATT AAA GTT TTA AAC AAG ACC CAG GAG AA - #C GGG ACC TGC ACC TTG          540                                                                       Glu Ile Lys Val Leu Asn Lys Thr Gln Glu As - #n Gly Thr Cys Thr Leu           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - ATA CTG GGC TGC ACA GTG GAG AAG GGG GAC CA - #T GTG GCT TAC AGC        TGG      588                                                                    Ile Leu Gly Cys Thr Val Glu Lys Gly Asp Hi - #s Val Ala Tyr Ser Trp                          165  - #               170  - #               175              - - AGT GAA AAG GCG GGC ACC CAC CCA CTG AAC CC - #A GCC AAC AGC TCC CAC          636                                                                       Ser Glu Lys Ala Gly Thr His Pro Leu Asn Pr - #o Ala Asn Ser Ser His                       180      - #           185      - #           190                  - - CTC CTG TCC CTC ACC CTC GGC CCC CAG CAT GC - #T GAC AAT ATC TAC ATC          684                                                                       Leu Leu Ser Leu Thr Leu Gly Pro Gln His Al - #a Asp Asn Ile Tyr Ile                   195          - #       200          - #       205                      - - TGC ACC GTG AGC AAC CCT ATC AGC AAC AAT TC - #C CAG ACC TTC AGC CCG          732                                                                       Cys Thr Val Ser Asn Pro Ile Ser Asn Asn Se - #r Gln Thr Phe Ser Pro               210              - #   215              - #   220                          - - TGG CCC GGA TGC AGG ACA GAC CCC TCA GGT AA - #A ACG AAC CAT TAC CAG          780                                                                       Trp Pro Gly Cys Arg Thr Asp Pro Ser Gly Ly - #s Thr Asn His Tyr Gln           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - ACA ACA GTG GAA AAA AAA AGC CTT ACG ATC TA - #T GCC CAA GTC CAG        AAA      828                                                                    Thr Thr Val Glu Lys Lys Ser Leu Thr Ile Ty - #r Ala Gln Val Gln Lys                          245  - #               250  - #               255              - - CCA GGT CCT CTT CAG AAG AAA CTT GAC TCC TT - #C CCA GCT CAG GAC CCT          876                                                                       Pro Gly Pro Leu Gln Lys Lys Leu Asp Ser Ph - #e Pro Ala Gln Asp Pro                       260      - #           265      - #           270                  - - TGC ACC ACC ATA TAT GTT GCT GCC ACA GAG CC - #T GTC CCA GAG TCT GTC          924                                                                       Cys Thr Thr Ile Tyr Val Ala Ala Thr Glu Pr - #o Val Pro Glu Ser Val                   275          - #       280          - #       285                      - - CAG GAA ACA AAT TCC ATC ACA GTC TAT GCT AG - #T GTG ACA CTT CCA GAG          972                                                                       Gln Glu Thr Asn Ser Ile Thr Val Tyr Ala Se - #r Val Thr Leu Pro Glu               290              - #   295              - #   300                          - - AGC TGACACCAGA GACCAACAAA GGGACTTTCT GAAGGAAAAT GGAAA - #                  1020                                                                        Ser                                                                           305                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 305 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Met Asp Pro Lys Gly Leu Leu Ser Leu Thr Ph - #e Val Leu Phe Leu Ser        1               5 - #                 10 - #                 15              - - Leu Ala Phe Gly Ala Ser Tyr Gly Thr Gly Gl - #y Arg Met Met Asn Cys                   20     - #             25     - #             30                  - - Pro Lys Ile Leu Arg Gln Leu Gly Ser Lys Va - #l Leu Leu Pro Leu Thr               35         - #         40         - #         45                      - - Tyr Glu Arg Ile Asn Lys Ser Met Asn Lys Se - #r Ile His Ile Val Val           50             - #     55             - #     60                          - - Thr Met Ala Lys Ser Leu Glu Asn Ser Val Gl - #u Asn Lys Ile Val Ser       65                 - # 70                 - # 75                 - # 80       - - Leu Asp Pro Ser Glu Ala Gly Pro Pro Arg Ty - #r Leu Gly Asp Arg Tyr                       85 - #                 90 - #                 95              - - Lys Phe Tyr Leu Glu Asn Leu Thr Leu Gly Il - #e Arg Glu Ser Arg Lys                  100      - #           105      - #           110                  - - Glu Asp Glu Gly Trp Tyr Leu Met Thr Leu Gl - #u Lys Asn Val Ser Val              115          - #       120          - #       125                      - - Gln Arg Phe Cys Leu Gln Leu Arg Leu Tyr Gl - #u Gln Val Ser Thr Pro          130              - #   135              - #   140                          - - Glu Ile Lys Val Leu Asn Lys Thr Gln Glu As - #n Gly Thr Cys Thr Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ile Leu Gly Cys Thr Val Glu Lys Gly Asp Hi - #s Val Ala Tyr Ser        Trp                                                                                             165  - #               170  - #               175             - - Ser Glu Lys Ala Gly Thr His Pro Leu Asn Pr - #o Ala Asn Ser Ser His                  180      - #           185      - #           190                  - - Leu Leu Ser Leu Thr Leu Gly Pro Gln His Al - #a Asp Asn Ile Tyr Ile              195          - #       200          - #       205                      - - Cys Thr Val Ser Asn Pro Ile Ser Asn Asn Se - #r Gln Thr Phe Ser Pro          210              - #   215              - #   220                          - - Trp Pro Gly Cys Arg Thr Asp Pro Ser Gly Ly - #s Thr Asn His Tyr Gln      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Thr Thr Val Glu Lys Lys Ser Leu Thr Ile Ty - #r Ala Gln Val Gln        Lys                                                                                             245  - #               250  - #               255             - - Pro Gly Pro Leu Gln Lys Lys Leu Asp Ser Ph - #e Pro Ala Gln Asp Pro                  260      - #           265      - #           270                  - - Cys Thr Thr Ile Tyr Val Ala Ala Thr Glu Pr - #o Val Pro Glu Ser Val              275          - #       280          - #       285                      - - Gln Glu Thr Asn Ser Ile Thr Val Tyr Ala Se - #r Val Thr Leu Pro Glu          290              - #   295              - #   300                          - - Ser                                                                      305                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1079 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 153..1073                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - GGACTCTGTT CCTGTCTTTC TGTCTATCTT CTTCCCAAGG CAGGCTATTG CT -             #TTCTGTTT     60                                                                 - - AGAAGTATCA GGGCTATGAG AAAAGGTATT TGAGAAAGAA AAAGCCAAGC AA -            #GAAGTGGA    120                                                                 - - CTTTGGACTG CCTGTGTGAG TGGGGTGGGC GC ATG ATG AAC TGC - # CCA AAG        ATT      173                                                                                      - #                  - #Met Met Asn Cys Pro Lys Ile                          - #                  - #  1               5                  - - CTC CGG CAG TTG GGA AGC AAA GTG CTG CTG CC - #C CTG ACA TAT GAA AGG          221                                                                       Leu Arg Gln Leu Gly Ser Lys Val Leu Leu Pr - #o Leu Thr Tyr Glu Arg                    10         - #         15         - #         20                      - - ATA AAT AAG AGC ATG AAC AAA AGC ATC CAC AT - #T GTC GTC ACA ATG GCA          269                                                                       Ile Asn Lys Ser Met Asn Lys Ser Ile His Il - #e Val Val Thr Met Ala                25             - #     30             - #     35                          - - AAA TCA CTG GAG AAC AGT GTC GAG AAC AAA AT - #A GTG TCT CTT GAT CCA          317                                                                       Lys Ser Leu Glu Asn Ser Val Glu Asn Lys Il - #e Val Ser Leu Asp Pro            40                 - # 45                 - # 50                 - # 55       - - TCC GAA GCA GGC CCT CCA CGT TAT CTA GGA GA - #T CGC TAC AAG TTT TAT          365                                                                       Ser Glu Ala Gly Pro Pro Arg Tyr Leu Gly As - #p Arg Tyr Lys Phe Tyr                            60 - #                 65 - #                 70              - - CTG GAG AAT CTC ACC CTG GGG ATA CGG GAA AG - #C AGG AAG GAG GAT GAG          413                                                                       Leu Glu Asn Leu Thr Leu Gly Ile Arg Glu Se - #r Arg Lys Glu Asp Glu                        75     - #             80     - #             85                  - - GGA TGG TAC CTT ATG ACC CTG GAG AAA AAT GT - #T TCA GTT CAG CGC TTT          461                                                                       Gly Trp Tyr Leu Met Thr Leu Glu Lys Asn Va - #l Ser Val Gln Arg Phe                    90         - #         95         - #        100                      - - TGC CTG CAG TTG AGG CTT TAT GAG CAG GTC TC - #C ACT CCA GAA ATT AAA          509                                                                       Cys Leu Gln Leu Arg Leu Tyr Glu Gln Val Se - #r Thr Pro Glu Ile Lys               105              - #   110              - #   115                          - - GTT TTA AAC AAG ACC CAG GAG AAC GGG ACC TG - #C ACC TTG ATA CTG GGC          557                                                                       Val Leu Asn Lys Thr Gln Glu Asn Gly Thr Cy - #s Thr Leu Ile Leu Gly           120                 1 - #25                 1 - #30                 1 -      #35                                                                              - - TGC ACA GTG GAG AAG GGG GAC CAT GTG GCT TA - #C AGC TGG AGT GAA        AAG      605                                                                    Cys Thr Val Glu Lys Gly Asp His Val Ala Ty - #r Ser Trp Ser Glu Lys                          140  - #               145  - #               150              - - GCG GGC ACC CAC CCA CTG AAC CCA GCC AAC AG - #C TCC CAC CTC CTG TCC          653                                                                       Ala Gly Thr His Pro Leu Asn Pro Ala Asn Se - #r Ser His Leu Leu Ser                       155      - #           160      - #           165                  - - CTC ACC CTC GGC CCC CAG CAT GCT GAC AAT AT - #C TAC ATC TGC ACC GTG          701                                                                       Leu Thr Leu Gly Pro Gln His Ala Asp Asn Il - #e Tyr Ile Cys Thr Val                   170          - #       175          - #       180                      - - AGC AAC CCT ATC AGC AAC AAT TCC CAG ACC TT - #C AGC CCG TGG CCC GGA          749                                                                       Ser Asn Pro Ile Ser Asn Asn Ser Gln Thr Ph - #e Ser Pro Trp Pro Gly               185              - #   190              - #   195                          - - TGC AGG ACA GAC CCC TCA GAA ACA AAA CCA TG - #G GCA GTG TAT GCT GGG          797                                                                       Cys Arg Thr Asp Pro Ser Glu Thr Lys Pro Tr - #p Ala Val Tyr Ala Gly           200                 2 - #05                 2 - #10                 2 -      #15                                                                              - - CTG TTA GGG GGT GTC ATC ATG ATT CTC ATC AT - #G GTG GTA ATA CTA        CAG      845                                                                    Leu Leu Gly Gly Val Ile Met Ile Leu Ile Me - #t Val Val Ile Leu Gln                          220  - #               225  - #               230              - - TTG AGA AGA AGA GGT AAA ACG AAC CAT TAC CA - #G ACA ACA GTG GAA AAA          893                                                                       Leu Arg Arg Arg Gly Lys Thr Asn His Tyr Gl - #n Thr Thr Val Glu Lys                       235      - #           240      - #           245                  - - AAA AGC CTT ACG ATC TAT GCC CAA GTC CAG AA - #A CCA GGT CCT CTT CAG          941                                                                       Lys Ser Leu Thr Ile Tyr Ala Gln Val Gln Ly - #s Pro Gly Pro Leu Gln                   250          - #       255          - #       260                      - - AAG AAA CTT GAC TCC TTC CCA GCT CAG GAC CC - #T TGC ACC ACC ATA TAT          989                                                                       Lys Lys Leu Asp Ser Phe Pro Ala Gln Asp Pr - #o Cys Thr Thr Ile Tyr               265              - #   270              - #   275                          - - GTT GCT GCC ACA GAG CCT GTC CCA GAG TCT GT - #C CAG GAA ACA AAT TCC         1037                                                                       Val Ala Ala Thr Glu Pro Val Pro Glu Ser Va - #l Gln Glu Thr Asn Ser           280                 2 - #85                 2 - #90                 2 -      #95                                                                              - - ATC ACA GTC TAT GCT AGT GTG ACA CTT CCA GA - #G AGC TGACAC                  - #1079                                                                   Ile Thr Val Tyr Ala Ser Val Thr Leu Pro Gl - #u Ser                                           300  - #               305                                     - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 307 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Met Met Asn Cys Pro Lys Ile Leu Arg Gln Le - #u Gly Ser Lys Val Leu        1               5 - #                 10 - #                 15              - - Leu Pro Leu Thr Tyr Glu Arg Ile Asn Lys Se - #r Met Asn Lys Ser Ile                   20     - #             25     - #             30                  - - His Ile Val Val Thr Met Ala Lys Ser Leu Gl - #u Asn Ser Val Glu Asn               35         - #         40         - #         45                      - - Lys Ile Val Ser Leu Asp Pro Ser Glu Ala Gl - #y Pro Pro Arg Tyr Leu           50             - #     55             - #     60                          - - Gly Asp Arg Tyr Lys Phe Tyr Leu Glu Asn Le - #u Thr Leu Gly Ile Arg       65                 - # 70                 - # 75                 - # 80       - - Glu Ser Arg Lys Glu Asp Glu Gly Trp Tyr Le - #u Met Thr Leu Glu Lys                       85 - #                 90 - #                 95              - - Asn Val Ser Val Gln Arg Phe Cys Leu Gln Le - #u Arg Leu Tyr Glu Gln                  100      - #           105      - #           110                  - - Val Ser Thr Pro Glu Ile Lys Val Leu Asn Ly - #s Thr Gln Glu Asn Gly              115          - #       120          - #       125                      - - Thr Cys Thr Leu Ile Leu Gly Cys Thr Val Gl - #u Lys Gly Asp His Val          130              - #   135              - #   140                          - - Ala Tyr Ser Trp Ser Glu Lys Ala Gly Thr Hi - #s Pro Leu Asn Pro Ala      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asn Ser Ser His Leu Leu Ser Leu Thr Leu Gl - #y Pro Gln His Ala        Asp                                                                                             165  - #               170  - #               175             - - Asn Ile Tyr Ile Cys Thr Val Ser Asn Pro Il - #e Ser Asn Asn Ser Gln                  180      - #           185      - #           190                  - - Thr Phe Ser Pro Trp Pro Gly Cys Arg Thr As - #p Pro Ser Glu Thr Lys              195          - #       200          - #       205                      - - Pro Trp Ala Val Tyr Ala Gly Leu Leu Gly Gl - #y Val Ile Met Ile Leu          210              - #   215              - #   220                          - - Ile Met Val Val Ile Leu Gln Leu Arg Arg Ar - #g Gly Lys Thr Asn His      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Tyr Gln Thr Thr Val Glu Lys Lys Ser Leu Th - #r Ile Tyr Ala Gln        Val                                                                                             245  - #               250  - #               255             - - Gln Lys Pro Gly Pro Leu Gln Lys Lys Leu As - #p Ser Phe Pro Ala Gln                  260      - #           265      - #           270                  - - Asp Pro Cys Thr Thr Ile Tyr Val Ala Ala Th - #r Glu Pro Val Pro Glu              275          - #       280          - #       285                      - - Ser Val Gln Glu Thr Asn Ser Ile Thr Val Ty - #r Ala Ser Val Thr Leu          290              - #   295              - #   300                          - - Pro Glu Ser                                                              305                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1200 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 61..1089                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - TCCTGCCGAG CTGAGCTGAG CTGAGCTCAC AGCTGGGACC CTGTCTGCGA TT -             #GCTGGCTA     60                                                                 - - ATG GAT CCC AAA GGA TCC CTT TCC TGG AGA AT - #A CTT CTG TTT CTC        TCC      108                                                                    Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Il - #e Leu Leu Phe Leu Ser            1               5 - #                 10 - #                 15              - - CTG GCT TTT GAG TTG AGC TAC GGA ACA GGT GG - #A GGT GTG ATG GAT TGC          156                                                                       Leu Ala Phe Glu Leu Ser Tyr Gly Thr Gly Gl - #y Gly Val Met Asp Cys                        20     - #             25     - #             30                  - - CCA GTG ATT CTC CAG AAG CTG GGA CAG GAC AC - #G TGG CTG CCC CTG ACG          204                                                                       Pro Val Ile Leu Gln Lys Leu Gly Gln Asp Th - #r Trp Leu Pro Leu Thr                    35         - #         40         - #         45                      - - AAT GAA CAT CAG ATA AAT AAG AGC GTG AAC AA - #A AGT GTC CGC ATC CTC          252                                                                       Asn Glu His Gln Ile Asn Lys Ser Val Asn Ly - #s Ser Val Arg Ile Leu                50             - #     55             - #     60                          - - GTC ACC ATG GCG ACG TCC CCA GGA AGC AAA TC - #C AAC AAG AAA ATT GTG          300                                                                       Val Thr Met Ala Thr Ser Pro Gly Ser Lys Se - #r Asn Lys Lys Ile Val            65                 - # 70                 - # 75                 - # 80       - - TCT TTT GAT CTC TCT AAA GGG AGC TAT CCA GA - #T CAC CTG GAG GAT GGC          348                                                                       Ser Phe Asp Leu Ser Lys Gly Ser Tyr Pro As - #p His Leu Glu Asp Gly                            85 - #                 90 - #                 95              - - TAC CAC TTT CAA TCG AAA AAC CTG AGC CTG AA - #G ATC CTC GGG AAC AGG          396                                                                       Tyr His Phe Gln Ser Lys Asn Leu Ser Leu Ly - #s Ile Leu Gly Asn Arg                       100      - #           105      - #           110                  - - CGG GAG AGT GAA GGA TGG TAC TTG GTG AGC GT - #G GAG GAG AAC GTT TCT          444                                                                       Arg Glu Ser Glu Gly Trp Tyr Leu Val Ser Va - #l Glu Glu Asn Val Ser                   115          - #       120          - #       125                      - - GTT CAG CAA TTC TGC AAG CAG CTG AAG CTT TA - #T GAA CAG GTC TCC CCT          492                                                                       Val Gln Gln Phe Cys Lys Gln Leu Lys Leu Ty - #r Glu Gln Val Ser Pro               130              - #   135              - #   140                          - - CCA GAG ATT AAA GTG CTA AAC AAA ACC CAG GA - #G AAC GAG AAT GGG ACC          540                                                                       Pro Glu Ile Lys Val Leu Asn Lys Thr Gln Gl - #u Asn Glu Asn Gly Thr           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - TGC AGC TTG CTG TTG GCC TGC ACA GTG AAG AA - #A GGG GAC CAT GTG        ACT      588                                                                    Cys Ser Leu Leu Leu Ala Cys Thr Val Lys Ly - #s Gly Asp His Val Thr                          165  - #               170  - #               175              - - TAC AGC TGG AGT GAT GAG GCA GGC ACC CAC CT - #G CTG AGC CGA GCC AAC          636                                                                       Tyr Ser Trp Ser Asp Glu Ala Gly Thr His Le - #u Leu Ser Arg Ala Asn                       180      - #           185      - #           190                  - - CGC TCC CAC CTC CTG CAC ATC ACT CTT AGC AA - #C CAG CAT CAA GAC AGC          684                                                                       Arg Ser His Leu Leu His Ile Thr Leu Ser As - #n Gln His Gln Asp Ser                   195          - #       200          - #       205                      - - ATC TAC AAC TGC ACC GCA AGC AAC CCT GTC AG - #C AGT ATC TCT AGG ACC          732                                                                       Ile Tyr Asn Cys Thr Ala Ser Asn Pro Val Se - #r Ser Ile Ser Arg Thr               210              - #   215              - #   220                          - - TTC AAC CTA TCA TCG CAA GCA TGC AAG CAG GA - #A TCC TCC TCA GAA TCG          780                                                                       Phe Asn Leu Ser Ser Gln Ala Cys Lys Gln Gl - #u Ser Ser Ser Glu Ser           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - AGT CCA TGG ATG CAA TAT ACT CTT GTA CCA CT - #G GGG GTC GTT ATA        ATC      828                                                                    Ser Pro Trp Met Gln Tyr Thr Leu Val Pro Le - #u Gly Val Val Ile Ile                          245  - #               250  - #               255              - - TTC ATC CTG GTT TTC ACG GCA ATA ATA ATG AT - #G AAA AGA CAA GGT AAA          876                                                                       Phe Ile Leu Val Phe Thr Ala Ile Ile Met Me - #t Lys Arg Gln Gly Lys                       260      - #           265      - #           270                  - - TCA AAT CAC TGC CAG CCA CCA GTG GAA GAA AA - #A AGC CTT ACT ATT TAT          924                                                                       Ser Asn His Cys Gln Pro Pro Val Glu Glu Ly - #s Ser Leu Thr Ile Tyr                   275          - #       280          - #       285                      - - GCC CAA GTA CAG AAA TCA GGG CCT CAA GAG AA - #G AAA CTT CAT GAT GCC          972                                                                       Ala Gln Val Gln Lys Ser Gly Pro Gln Glu Ly - #s Lys Leu His Asp Ala               290              - #   295              - #   300                          - - CTA ACA GAT CAG GAC CCC TGC ACA ACC ATT TA - #T GTG GCT GCC ACA GAG         1020                                                                       Leu Thr Asp Gln Asp Pro Cys Thr Thr Ile Ty - #r Val Ala Ala Thr Glu           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - CCT GCC CCA GAG TCT GTC CAG GAA CCA AAC CC - #C ACC ACA GTT TAT        GCC     1068                                                                    Pro Ala Pro Glu Ser Val Gln Glu Pro Asn Pr - #o Thr Thr Val Tyr Ala                          325  - #               330  - #               335              - - AGT GTG ACA CTG CCA GAG AGC TGACCCATAT ACCCAGTGA - #A AGGACTTTTT            1119                                                                       Ser Val Thr Leu Pro Glu Ser                                                               340                                                                - - GAAGGAGGAT AGAAGAACCA AAATCCACAC TGAACTGGAC CCCGGGGTCC AA -             #GTTCTCTG   1179                                                                 - - TGACAGAAAC TGCACATCTG T           - #                  - #                    1200                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 343 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Il - #e Leu Leu Phe Leu Ser        1               5 - #                 10 - #                 15              - - Leu Ala Phe Glu Leu Ser Tyr Gly Thr Gly Gl - #y Gly Val Met Asp Cys                   20     - #             25     - #             30                  - - Pro Val Ile Leu Gln Lys Leu Gly Gln Asp Th - #r Trp Leu Pro Leu Thr               35         - #         40         - #         45                      - - Asn Glu His Gln Ile Asn Lys Ser Val Asn Ly - #s Ser Val Arg Ile Leu           50             - #     55             - #     60                          - - Val Thr Met Ala Thr Ser Pro Gly Ser Lys Se - #r Asn Lys Lys Ile Val       65                 - # 70                 - # 75                 - # 80       - - Ser Phe Asp Leu Ser Lys Gly Ser Tyr Pro As - #p His Leu Glu Asp Gly                       85 - #                 90 - #                 95              - - Tyr His Phe Gln Ser Lys Asn Leu Ser Leu Ly - #s Ile Leu Gly Asn Arg                  100      - #           105      - #           110                  - - Arg Glu Ser Glu Gly Trp Tyr Leu Val Ser Va - #l Glu Glu Asn Val Ser              115          - #       120          - #       125                      - - Val Gln Gln Phe Cys Lys Gln Leu Lys Leu Ty - #r Glu Gln Val Ser Pro          130              - #   135              - #   140                          - - Pro Glu Ile Lys Val Leu Asn Lys Thr Gln Gl - #u Asn Glu Asn Gly Thr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Cys Ser Leu Leu Leu Ala Cys Thr Val Lys Ly - #s Gly Asp His Val        Thr                                                                                             165  - #               170  - #               175             - - Tyr Ser Trp Ser Asp Glu Ala Gly Thr His Le - #u Leu Ser Arg Ala Asn                  180      - #           185      - #           190                  - - Arg Ser His Leu Leu His Ile Thr Leu Ser As - #n Gln His Gln Asp Ser              195          - #       200          - #       205                      - - Ile Tyr Asn Cys Thr Ala Ser Asn Pro Val Se - #r Ser Ile Ser Arg Thr          210              - #   215              - #   220                          - - Phe Asn Leu Ser Ser Gln Ala Cys Lys Gln Gl - #u Ser Ser Ser Glu Ser      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ser Pro Trp Met Gln Tyr Thr Leu Val Pro Le - #u Gly Val Val Ile        Ile                                                                                             245  - #               250  - #               255             - - Phe Ile Leu Val Phe Thr Ala Ile Ile Met Me - #t Lys Arg Gln Gly Lys                  260      - #           265      - #           270                  - - Ser Asn His Cys Gln Pro Pro Val Glu Glu Ly - #s Ser Leu Thr Ile Tyr              275          - #       280          - #       285                      - - Ala Gln Val Gln Lys Ser Gly Pro Gln Glu Ly - #s Lys Leu His Asp Ala          290              - #   295              - #   300                          - - Leu Thr Asp Gln Asp Pro Cys Thr Thr Ile Ty - #r Val Ala Ala Thr Glu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Pro Ala Pro Glu Ser Val Gln Glu Pro Asn Pr - #o Thr Thr Val Tyr        Ala                                                                                             325  - #               330  - #               335             - - Ser Val Thr Leu Pro Glu Ser                                                          340                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1140 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 61..1047                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - TCCTGCCGAG CTGAGCTGAG CTGAGCTCAC AGCTGGGACC CTGTCTGCGA TT -             #GCTGGCTA     60                                                                 - - ATG GAT CCC AAA GGA TCC CTT TCC TGG AGA AT - #A CTT CTG TTT CTC        TCC      108                                                                    Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Il - #e Leu Leu Phe Leu Ser            1               5 - #                 10 - #                 15              - - CTG GCT TTT GAG TTG AGC TAC GGA ACA GGT GG - #A GGT GTG ATG GAT TGC          156                                                                       Leu Ala Phe Glu Leu Ser Tyr Gly Thr Gly Gl - #y Gly Val Met Asp Cys                        20     - #             25     - #             30                  - - CCA GTG ATT CTC CAG AAG CTG GGA CAG GAC AC - #G TGG CTG CCC CTG ACG          204                                                                       Pro Val Ile Leu Gln Lys Leu Gly Gln Asp Th - #r Trp Leu Pro Leu Thr                    35         - #         40         - #         45                      - - AAT GAA CAT CAG ATA AAT AAG AGC GTG AAC AA - #A AGT GTC CGC ATC CTC          252                                                                       Asn Glu His Gln Ile Asn Lys Ser Val Asn Ly - #s Ser Val Arg Ile Leu                50             - #     55             - #     60                          - - GTC ACC ATG GCG ACG TCC CCA GGA AGC AAA TC - #C AAC AAG AAA ATT GTG          300                                                                       Val Thr Met Ala Thr Ser Pro Gly Ser Lys Se - #r Asn Lys Lys Ile Val            65                 - # 70                 - # 75                 - # 80       - - TCT TTT GAT CTC TCT AAA GGG AGC TAT CCA GA - #T CAC CTG GAG GAT GGC          348                                                                       Ser Phe Asp Leu Ser Lys Gly Ser Tyr Pro As - #p His Leu Glu Asp Gly                            85 - #                 90 - #                 95              - - TAC CAC TTT CAA TCG AAA AAC CTG AGC CTG AA - #G ATC CTC GGG AAC AGG          396                                                                       Tyr His Phe Gln Ser Lys Asn Leu Ser Leu Ly - #s Ile Leu Gly Asn Arg                       100      - #           105      - #           110                  - - CGG GAG AGT GAA GGA TGG TAC TTG GTG AGC GT - #G GAG GAG AAC GTT TCT          444                                                                       Arg Glu Ser Glu Gly Trp Tyr Leu Val Ser Va - #l Glu Glu Asn Val Ser                   115          - #       120          - #       125                      - - GTT CAG CAA TTC TGC AAG CAG CTG AAG CTT TA - #T GAA CAG GTC TCC CCT          492                                                                       Val Gln Gln Phe Cys Lys Gln Leu Lys Leu Ty - #r Glu Gln Val Ser Pro               130              - #   135              - #   140                          - - CCA GAG ATT AAA GTG CTA AAC AAA ACC CAG GA - #G AAC GAG AAT GGG ACC          540                                                                       Pro Glu Ile Lys Val Leu Asn Lys Thr Gln Gl - #u Asn Glu Asn Gly Thr           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - TGC AGC TTG CTG TTG GCC TGC ACA GTG AAG AA - #A GGG GAC CAT GTG        ACT      588                                                                    Cys Ser Leu Leu Leu Ala Cys Thr Val Lys Ly - #s Gly Asp His Val Thr                          165  - #               170  - #               175              - - TAC AGC TGG AGT GAT GAG GCA GGC ACC CAC CT - #G CTG AGC CGA GCC AAC          636                                                                       Tyr Ser Trp Ser Asp Glu Ala Gly Thr His Le - #u Leu Ser Arg Ala Asn                       180      - #           185      - #           190                  - - CGC TCC CAC CTC CTG CAC ATC ACT CTT AGC AA - #C CAG CAT CAA GAC AGC          684                                                                       Arg Ser His Leu Leu His Ile Thr Leu Ser As - #n Gln His Gln Asp Ser                   195          - #       200          - #       205                      - - ATC TAC AAC TGC ACC GCA AGC AAC CCT GTC AG - #C AGT ATC TCT AGG ACC          732                                                                       Ile Tyr Asn Cys Thr Ala Ser Asn Pro Val Se - #r Ser Ile Ser Arg Thr               210              - #   215              - #   220                          - - TTC AAC CTA TCA TCG CAA GCA TGC AAG CAG GA - #A TCC TCC TCA GAA TCG          780                                                                       Phe Asn Leu Ser Ser Gln Ala Cys Lys Gln Gl - #u Ser Ser Ser Glu Ser           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - AGT CCA TGG ATG CAA TAT ACT CTT GTA CCA CT - #G GGG GTC GTT ATA        ATC      828                                                                    Ser Pro Trp Met Gln Tyr Thr Leu Val Pro Le - #u Gly Val Val Ile Ile                          245  - #               250  - #               255              - - TTC ATC CTG GTT TTC ACG GCA ATA ATA ATG AT - #G AAA AGA CAA GGT AAA          876                                                                       Phe Ile Leu Val Phe Thr Ala Ile Ile Met Me - #t Lys Arg Gln Gly Lys                       260      - #           265      - #           270                  - - TCA AAT CAC TGC CAG CCA CCA GTG GAA GAA AA - #A AGC CTT ACT ATT TAT          924                                                                       Ser Asn His Cys Gln Pro Pro Val Glu Glu Ly - #s Ser Leu Thr Ile Tyr                   275          - #       280          - #       285                      - - GCC CAA GTA CAG AAA TCA GGG GTA CGT TCT AT - #G CCT CAC CTT GCG GGA          972                                                                       Ala Gln Val Gln Lys Ser Gly Val Arg Ser Me - #t Pro His Leu Ala Gly               290              - #   295              - #   300                          - - GTG TCT GTC ATA TTT CGC ACA GGA TTT CTG AT - #A GCT GCC TTG CAC ACA         1020                                                                       Val Ser Val Ile Phe Arg Thr Gly Phe Leu Il - #e Ala Ala Leu His Thr           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - ACC ATG GTC CTG CAG GGA CTC CTA GAG TAGATGAAC - #T TAAGAAAGCA               1067                                                                      Thr Met Val Leu Gln Gly Leu Leu Glu                                                           325                                                            - - GAAAAGTCAA GAACAAGAGC TCCCCCAGTG TCACTGACCC TTATATTGTT TG -             #AACTTGTA   1127                                                                 - - GAAAACAGTG ACA              - #                  - #                      - #    1140                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 329 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Il - #e Leu Leu Phe Leu Ser        1               5 - #                 10 - #                 15              - - Leu Ala Phe Glu Leu Ser Tyr Gly Thr Gly Gl - #y Gly Val Met Asp Cys                   20     - #             25     - #             30                  - - Pro Val Ile Leu Gln Lys Leu Gly Gln Asp Th - #r Trp Leu Pro Leu Thr               35         - #         40         - #         45                      - - Asn Glu His Gln Ile Asn Lys Ser Val Asn Ly - #s Ser Val Arg Ile Leu           50             - #     55             - #     60                          - - Val Thr Met Ala Thr Ser Pro Gly Ser Lys Se - #r Asn Lys Lys Ile Val       65                 - # 70                 - # 75                 - # 80       - - Ser Phe Asp Leu Ser Lys Gly Ser Tyr Pro As - #p His Leu Glu Asp Gly                       85 - #                 90 - #                 95              - - Tyr His Phe Gln Ser Lys Asn Leu Ser Leu Ly - #s Ile Leu Gly Asn Arg                  100      - #           105      - #           110                  - - Arg Glu Ser Glu Gly Trp Tyr Leu Val Ser Va - #l Glu Glu Asn Val Ser              115          - #       120          - #       125                      - - Val Gln Gln Phe Cys Lys Gln Leu Lys Leu Ty - #r Glu Gln Val Ser Pro          130              - #   135              - #   140                          - - Pro Glu Ile Lys Val Leu Asn Lys Thr Gln Gl - #u Asn Glu Asn Gly Thr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Cys Ser Leu Leu Leu Ala Cys Thr Val Lys Ly - #s Gly Asp His Val        Thr                                                                                             165  - #               170  - #               175             - - Tyr Ser Trp Ser Asp Glu Ala Gly Thr His Le - #u Leu Ser Arg Ala Asn                  180      - #           185      - #           190                  - - Arg Ser His Leu Leu His Ile Thr Leu Ser As - #n Gln His Gln Asp Ser              195          - #       200          - #       205                      - - Ile Tyr Asn Cys Thr Ala Ser Asn Pro Val Se - #r Ser Ile Ser Arg Thr          210              - #   215              - #   220                          - - Phe Asn Leu Ser Ser Gln Ala Cys Lys Gln Gl - #u Ser Ser Ser Glu Ser      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ser Pro Trp Met Gln Tyr Thr Leu Val Pro Le - #u Gly Val Val Ile        Ile                                                                                             245  - #               250  - #               255             - - Phe Ile Leu Val Phe Thr Ala Ile Ile Met Me - #t Lys Arg Gln Gly Lys                  260      - #           265      - #           270                  - - Ser Asn His Cys Gln Pro Pro Val Glu Glu Ly - #s Ser Leu Thr Ile Tyr              275          - #       280          - #       285                      - - Ala Gln Val Gln Lys Ser Gly Val Arg Ser Me - #t Pro His Leu Ala Gly          290              - #   295              - #   300                          - - Val Ser Val Ile Phe Arg Thr Gly Phe Leu Il - #e Ala Ala Leu His Thr      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Thr Met Val Leu Gln Gly Leu Leu Glu                                                      325                                                         __________________________________________________________________________

What is claimed is:
 1. A substantially pure or recombinant SLAMprotein:a) consisting of SEQ ID NO: 2,4, 6, 8, 10, or 12; b) encoded bya polynucleotide comprising the open reading frame of SEQ ID NO:1,3,5,7,9,or 11; or c) encoded by a naturally occurring allelic variantof a polynucleotide comprising the open reading frame of SEQ ID NO: 1,3, 5, 7, 9, or
 11. 2. The recombinant protein of claim 1, encoded by anaturally occurring allelic variant of a polynucleotide comprising theopen reading frame of SEQ ID NO: 1,3,5, or
 7. 3. A substantially pure orrecombinant SLAM protein which:a) has an apparent molecular weight 70 kDfor naturally glycosylated SLAM protein, as determined bySDS/polyacrylamide gel electrophoresis; and b) is found on immatureCD3^(low) CD4⁺ CD8⁺ fetal thymocytes or peripheral blood CD45RO^(high)memory T cells;wherein said SLAM protein has at least one of thefollowing properties: i) is rapidly induced on naive CD45RA⁺ cellsfollowing activation; ii) is a self-ligand; or iii) stimulatesproliferation of CD4⁺ T cell dones upon specific engagement with saidSLAM polypeptide.
 4. A substantially pure or recombinant polypeptidecomprising at least 12 contiguous amino acid residues identical to atleast 12 contiguous amino acid residues of said SLAM protein of claim 1.5. A substantially pure or recombinant polypeptide comprising at least16 contiguous amino acid residues identical to at least 16 contiguousamino acid residues of said SLAM protein of claim
 2. 6. A substantiallypure or recombinant polypeptide comprising at least 20 contiguous aminoacid residues identical to at least 20 contiguous amino acid residues ofsaid SLAM protein of claim
 1. 7. A substantially pure or recombinantpolypeptide comprising at least 30 contiguous amino acid residues thatare identical to at least 30 contiguous amino acid residues of said SLAMprotein of claim
 1. 8. A substantially pure or recombinant polypeptidecomprising the intracellular domain, the extracellular domain, or themature form of said SLAM protein of claim
 1. 9. A substantially pure orrecombinant polypeptide comprising the intracellular domain, theextracellular domain, or the mature form of said SLAM protein of claim3.
 10. A substantially pure or recombinant polypeptide comprising animmunogenic peptide of said SLAM protein of claim
 1. 11. A substantiallypure or recombinant polypeptide comprising an immunogenic peptide ofsaid SLAM protein of claim
 3. 12. A substantially pure or recombinantpolypeptide comprising an immunogenic peptide of said polypeptide ofclaim
 4. 13. A substantially pure or recombinant pplypeptide comprisingan immunogenic peptide of said polypeptide of claim
 7. 14. A fusionprotein comprising said polypeptide of claim 4 and:a) a detection orpurification tag selected from the group consisting of a FLAG, His6, andimmunoglobulin peptide; b) a carrier protein selected from the groupconsisting of keyhole limpet hemocyanin, bovine serum albumin, andtetanus toxoid; or c) another peptide selected from the group consistingof luciferase, bacterial β-galactosidase, trpE, protein A, β-lactamase,alpha amylase, alcohol dehydrogenase, and yeast alpha mating factor. 15.A fusion protein comprising said polypeptide of claim 7 and:a) adetection or purification tag selected from the group consisting of aFLAG, His6, and immunoglobulin peptide; b) a carrier protein selectedfrom the group consisting of keyhole limpet hemocyanin, bovine serumalbumin, and tetanus toxoid; or c) another peptide selected from thegroup consisting of luciferase, bacterial β-galactosidase, trpE, proteinA, β-lactamase, alpha amylase, alcohol dehydrogenase, and yeast alphamating factor.
 16. A fusion protein comprising said polypeptide of claim11 and:a) a detection or purification tag selected from the groupconsisting of a FLAG, His6, and immunoglobulin peptide; b) a carrierprotein selected from the group consisting of keyhole limpet hemocyanin,bovine serum albumin, the tetanus toxoid; or c) another peptide selectedfrom the group consisting of luciferase, bacterial β-galactosidase,trpE, protein A, β-lactamase, alpha amylase, alcohol dehydrogenase, andyeast alpha mating factor.
 17. A composition comprising said polypeptideof claim 4, that is:a) in a pharmaceutically acceptable carrier; b) in asterile composition; c) in a buffered solution; or d) in an aqueoussuspension.
 18. The composition of claim 17, in water, saline, buffer,or an aqueous solution.
 19. A composition comprising said polypeptide ofclaim 7, that is:a) in a pharmaceutically acceptable carrier; b) in asterile composition; c) in a buffered solution; or d) in an aqueoussuspension.
 20. A composition comprising said polypeptide of claim 11,that is:a) in a pharmaceutically acceptable carrier; b) in a sterilecomposition; c) in a buffered solution; or d) in an aqueous suspension.21. A polypeptide of claim 4, that is:a) denatured; b) immunopurified;c) attached to a solid substrate; d) detectably labeled; or e)chemically synthesized.
 22. A polypeptide of claim 11, that is:a)denatured; b) immunopurified; c) attached to a solid substrate; d)detectably labeled; or e) chemically synthesized.
 23. A kit comprisingsaid protein of claim 1, and:a) a compartment comprising said protein;or b) instructions for use or disposal of reagents in said kit.
 24. Akit comprising said polypeptide of claim 3, and:a) a compartmentcomprising said protein; or b) instructions for use or disposal ofreagents in said kit.
 25. A kit comprising said polypeptide of claim 4,and:a) a compartment comprising said polypeptide; or b) instructions foruse or disposal of reagents in said kit.
 26. A kit comprising saidpolypeptide of claim 7, and:a) a compartment comprising saidpolypeptide; or b) instructions for use or disposal of reagents in saidkit.
 27. A kit comprising said polypeptide of claim 11, and:a) acompartment comprising said polypeptide; or b) instructions for use ordisposal of reagents in said kit.
 28. The SLAM protein of claim 1,which:a) induces cytokine production by CD4⁺ T cell clones; b) reversesa phenotype of a Th2 cell clone to a Th0 T cell done; or c) has anapparent molecular weight of about 40 kD as determined by SDSpolyacrylamide gel electrophoresis, following N-glycanase treatment ofsaid SLAM protein.
 29. The SLAM protein of claim 3, which:a) inducescytokine production by CD4⁺ T cell clones; b) reverses a phenotype of aTh2 cell clone to a Th0 T cell clone; or c) has an apparent molecularweight of about 40 kD as determined by SDS polyacrylamide gelelectrophoresis, following N-glycanase treatment of said SLAM protein.30. The polypeptide of claim 9, wherein:a) said intracellular domain isfrom amino acid residue 28 to 230 of SEQ ID NO: 2; or b) saidextracellular domain is from amino acid residue 258 to 335 of SEQ ID NO:2.